Surface-modified semiconductive and metallic nanoparticles having enhanced dispersibility in aqueous media

ABSTRACT

Water-dispersible nanoparticles are prepared by applying a coating of a multiply amphipathic dispersant to the surface of a hydrophobic nanoparticle comprised of a semiconductive or metallic material. The multiply amphipathic dispersant has two or more hydrophobic regions and two or more hydrophilic regions, and is typically polymeric. Preferred polymeric dispersants are comprised of (1) a hydrophobic backbone with hydrophilic branches, (2) a hydrophilic backbone with hydrophobic branches, or (3) a backbone that may be either hydrophobic or hydrophilic, and substituted with both hydrophilic and hydrophobic branches. Monodisperse populations of water-dispersible nanoparticles are also provided, as are conjugates of the water-dispersible nanoparticles with affinity molecules such as peptides, oligonucleotides, and the like.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/841,237, filed Apr. 23, 2001, which claims priority to U.S.Provisional Application No. 60/240,216, filed Oct. 13, 2000. Thedisclosures of the aforementioned applications are incorporated byreference in their entireties.

TECHNICAL FIELD

[0002] This invention relates generally to surface-modifiednanoparticles, and more particularly relates to surface-modifiedsemiconductor and metal nanoparticles having enhanced dispersibility inaqueous media as well as superior colloidal and photophysical stability.The invention additionally relates to methods for making and using thenovel surface-modified nanoparticles. The invention finds utility in avariety of fields, including biology, analytical and combinatorialchemistry, medical diagnostics, and genetic analysis.

BACKGROUND

[0003] Semiconductor nanocrystals (also known as quantum dot particles)whose radii are smaller than the bulk exciton Bohr radius constitute aclass of materials intermediate between molecular and bulk forms ofmatter. Quantum confinement of both the electron and hole in all threedimensions leads to an increase in the effective band gap of thematerial with decreasing crystallite size. Consequently, both theoptical absorption and emission of semiconductor nanocrystals shift tothe blue (higher energies) as the size of the nanocrystals gets smaller.

[0004] Semiconductor nanocrystals are nanoparticles composed of aninorganic, crystalline semiconductive material and have uniquephotophysical, photochemical and nonlinear optical properties arisingfrom quantum size effects, and have therefore attracted a great deal ofattention for their potential applicability in a variety of contexts,e.g., as detectable labels in biological applications, and as usefulmaterials in the areas of photocatalysis, charge transfer devices, andanalytical chemistry. As a result of the increasing interest insemiconductor nanocrystals, there is now a fairly substantial body ofliterature pertaining to methods for manufacturing such nanocrystals.Broadly, these routes may be classified as involving preparation inglasses (see Ekimov et al. (1981) JETP Letters 34:345), aqueouspreparation (including preparation that involve use of inverse micelles,zeolites, Langmuir-Blodgett films, and chelating polymers; see Fendleret al. (1984) J. Chem. Society, Chemical Communications 90:90, andHenglein et al. (1984) Ber. Bunsenges. Phys. Chem. 88:969), and hightemperature pyrolysis of organometallic semiconductor precursormaterials (Murray et al. (1993) J. Am. Chem. Soc. 115:8706; Katari etal. (1994) J. Phys. Chem. 98:4109). The two former methods yieldparticles that have unacceptably low quantum yields for mostapplications, a high degree of polydispersity, poor colloidal stability,a high degree of internal defects, and poorly passivated surface trapsites. In addition, nanocrystals made by the first route are physicallyconfined to a glass matrix and cannot be further processed aftersynthesis.

[0005] To date, only the high temperature pyrolysis of organometallicreagents has yielded semiconductor nanocrystals that are internallydefect free, possess high band edge luminescence and no trappedemission, and exhibit near monodispersity. Additionally, this routegives the synthetic chemist a substantial degree of control over thesize of the particles prepared. See Murray et al. (1993), supra. Onedisadvantage of this method, however, is that the particles aresequestered in reverse micelles of coordinated, hydrophobic surfactantmolecules. As such, they are only dispersible in organic solvents suchas chloroform, dichloromethane, hexane, toluene, and pyridine. This isproblematic insofar as many applications that rely on the fluorescenceemission of the semiconductor nanocrystals require that the nanocrystalsbe water soluble or at least water dispersible.

[0006] Although some methods for rendering semiconductor nanocrystalswater dispersible have been reported, they are still problematic insofaras the treated semiconductor nanocrystals suffer from significantdisadvantages that limit their wide applicability. For example, Spanhelet al. (1987) J. Am. Chem. Soc. 109:5649, discloses a Cd(OH)₂-capped CdSsol; however, the photoluminescent properties of the sol were pHdependent. The sol could be prepared only in a very narrow pH range (pH8-10) and exhibited a narrow fluorescence band only at a pH of greaterthan 10. Such pH dependency greatly limits the usefulness of thematerial; in particular, it is not appropriate for use in biologicalsystems.

[0007] Other groups have replaced the organic passivating layer of thesemiconductor nanocrystal with water-soluble moieties; however, theresultant derivatized semiconductor nanocrystals are not highlyluminescent. Short chain thiols such as 2-mercaptoethanol and1-thio-glycerol have been used as stabilizers in the preparation ofwater-soluble CdTe nanocrystals. See, Rogach et al. (1996) Ber.Bunsenges. Phys. Chem. 100:1772 and Rajh et al. (1993) J. Phys. Chem.97:11999. Other more exotic capping compounds have been reported withsimilar results. See Coffer et al. (1992) Nanotechnology 3:69, whichdescribes the use of deoxyribonucleic acid (DNA) as a capping compound.In all of these systems, the coated semiconductor nanocrystals were notstable and photoluminescent properties degraded with time.

[0008] Thus, to use these high quantum yield materials in applicationsthat require an aqueous medium, one must find a way of changing thepolarity of the organic coating, thereby facilitating the transfer ofthese particles to water. A great deal of work has been conducted onsurface exchange reactions that seek to replace the oleophilichydrocarbon coating on the nanocrystal surface with a range ofbifunctional polar molecules wherein one functional group of the cappingmolecule bears some affinity for the surface of the nanocrystal, whilethe other functional group, by virtue of its ionizability or high degreeof hydration, renders the nanocrystal water soluble. For example,International Patent Publication No. WO 00/17655 to Bawendi et al.describes a method for rendering semiconductor nanocrystals waterdispersible wherein monomeric surfactants are used as dispersing agents,with the hydrophobic region of the surfactants promoting associationwith the nanocrystals, while the hydrophilic region has affinity for anaqueous medium and stabilizes an aqueous suspension of the nanocrystals.International Patent Publication No. WO 00/17656 to Bawendi et al.describes a similar method wherein monomeric compounds of formulaHS—(CH₂)_(n)—X, wherein n is preferably ≧10 and X is carboxylate orsulfonate, are used in place of the monomeric surfactants.

[0009] Kuno et al. (1997) J. Chem. Phys. 106:9869-9882, Mikulec,“Semiconductor Nanocrystal Colloids: Manganese Doped Cadmium Selenide,(Core) Shell Composites for Biological Labeling, and Highly FluorescentCadmium Telluride,” doctoral dissertation, Massachusetts Institute ofTechnology (September 1999), and International Patent Publication No. WO00/17656 to Bawendi et al., cited supra, give detailed descriptions ofsurface exchange reactions designed to improve the water dispersibilityof hydrophobic nanocrystals. In general, these references indicate that:exchange of the original hydrophobic surfactant layer on the nanocrystalsurface is never quite complete, with retention of only about 10% toabout 15% of the surfactant (even after multiple exchange reactions);although never quantitatively displaced, exchange of the originalphosphine/phosphine oxide surfactant layer with more polar ligandsresults in a substantial decrease in quantum yield that is neverentirely regained; once dispersed in water, the particles have limitedcolloidal stability; and attempts to carry out further chemistry withthese particles, such as linking them to biomolecules through theirpendant carboxyl functionalities, is highly irreproducible and dependenton the size of the nanocrystal.

[0010] Thus, there remains a need in the art for a reliable,reproducible method for rendering hydrophobic semiconductor nanocrystalsdispersible in aqueous media while preserving the quantum efficienciesof the original particles, maintaining colloidal stability, and avoidingor minimizing any change in particle size distribution. Ideally, such amethod would be useful not only with semiconductor nanoparticles, butalso with other types of nanoparticles having hydrophobic surfaces,e.g., semiconductive nanoparticles that are not necessarily crystallineand metallic nanoparticles that may or may not be surface-modified.

SUMMARY OF THE INVENTION

[0011] It is accordingly a primary object of the invention to addressthe aforementioned need in the art by providing surface-modifiednanoparticles having enhanced dispersibility in aqueous media, whereinthe nanoparticles are comprised of an inner core having a hydrophobicsurface and an outer layer of a multiply amphipathic dispersant.

[0012] It is still another object of the invention to provide suchsurface-modified nanoparticles wherein the inner core is composed of asemiconductive or metallic material.

[0013] It is yet another object of the invention to provide suchnanoparticles wherein the multiply amphipathic dispersant is a polymerhaving two or more hydrophobic regions and two or more hydrophilicregions.

[0014] It is a further object of the invention to provide a method forpreparing a population of the aforementioned water-dispersiblenanoparticles.

[0015] It is still a further object of the invention to provide acomposition composed of a nanoparticle conjugate, i.e., awater-dispersible nanoparticle as above, conjugated to an affinitymolecule that serves as the first member of a binding pair.

[0016] It is yet a further object of the invention to provide such acomposition wherein a second member of the binding pair is associatedwith the first member through either covalent or noncovalentinteraction.

[0017] It is an additional object of the invention to provide amonodisperse population of water-dispersible nanoparticles wherein thepopulation is characterized in that it exhibits no more than about a 10%rms deviation, preferably no more than about a 5% rms deviation, in thediameter of the inner core.

[0018] Additional objects, advantages, and novel features of theinvention will be set forth in part in the description which follows,and in part will become apparent to those skilled in the art uponexamination of the following, or may be learned by practice of theinvention.

[0019] In one aspect of the invention, then, a water-dispersiblenanoparticle is provided that is comprised of an inner core and an outerlayer of a multiply amphipathic dispersant, i.e., a compound having twoor more hydrophobic regions and two or more hydrophilic regions. Theinner core comprises a semiconductive or metallic material, preferablyan inorganic semiconductive material that is in a crystalline state.Generally, the inner core also comprises a hydrophobic passivating layeron the semiconductive or metallic material resulting from solventsand/or surfactants used in nanoparticle manufacture. The surface of theinner core is accordingly hydrophobic, and the hydrophobic regions ofthe dispersant thus have affinity for the core surface and attachthereto, while the hydrophilic regions of the dispersant extend outwardfrom the nanoparticle and provide for dispersibility in water. In apreferred embodiment, the dispersant is polymeric and has a plurality ofboth hydrophobic regions and hydrophilic regions, thus enhancing waterdispersibility of the nanoparticle as well as the dispersant's affinityfor the core surface. Particularly preferred dispersants arehyperbranched or dendritic polymers, which, relative to prior methodsthat involve monomeric dispersants, substantially increase the waterdispersibility and colloidal stability of the nanoparticles. In apreferred embodiment, the nanoparticles are luminescent semiconductivenanocrystals, and include an overcoating “shell” layer between the innercore and the multiply amphipathic outer layer to increase luminescenceefficiency. The shell material has a higher bandgap energy than thenanocrystal core, and should also have good conduction and valence bandoffset with respect to the nanocrystal core. Further, an “affinitymolecule,” i.e., one member of a binding pair, may be attached to theouter layer of the surface-modified molecule, providing a nanoparticle“conjugate” that is useful in detecting the presence or quantity oftarget molecules that comprise the second member of the binding pair.The affinity molecule may be, for example, a protein, anoligonucleotide, an enzyme inhibitor, a polysaccharide, or a smallmolecule having a molecular weight of less than about 1500 grams/Mol.

[0020] In a related aspect of the invention, then, a composition isprovided that is comprised of the aforementioned nanoparticle conjugatein association with the second member of the binding pair, wherein theassociation may involve either covalent or noncovalent interaction.

[0021] In another aspect of the invention, a monodisperse population ofsurface-modified nanoparticles is provided, comprising a plurality ofwater-dispersible nanoparticles each having an inner core comprised of asemiconductive or metallic material and, surrounding the inner core, anouter layer comprised of a multiply amphipathic dispersant as describedabove, wherein the population is characterized in that the nanoparticlesare of substantially the same size and shape, i.e., the populationexhibits no more than about a 10% rms deviation in the diameter of theinner core, preferably no more than about a 5% rms deviation in thediameter of the inner core. The narrow size distribution of amonodisperse population increases the “information density” that isobtainable as a result of the particles' luminescence, i.e., the numberof discrete luminescence emissions obtainable for a given nanoparticlecomposition.

[0022] In another aspect of the invention, a method is provided formaking the surface-modified nanoparticles described above. The methodinvolves (a) admixing (i) an amphipathic dispersant comprised of apolymer having two or more hydrophobic regions and two or morehydrophilic regions, with (ii) a plurality of hydrophobic nanoparticles,in (iii) a nonaqueous solvent, to provide an admixture of dispersant andnanoparticles in solution; (b) subjecting the admixture to conditionseffective to cause adsorption of the dispersant by the nanoparticles;and (c) transferring the dispersant-coated nanoparticles prepared instep (b) to an aqueous medium such as water or an aqueous buffer.

DETAILED DESCRIPTION OF THE INVENTION

[0023] I. Definitions:

[0024] Before describing the present invention in detail, it is to beunderstood that unless otherwise indicated this invention is not limitedto specific nanoparticle materials, amphipathic dispersants, ormanufacturing processes, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting.

[0025] It must be noted that, as used in this specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, “a dispersant” refers to a single dispersant as well as amixture of two or more dispersants, “a nanoparticle” encompasses notonly a single nanoparticle but also two or more nanoparticles, and thelike.

[0026] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0027] The term “amphipathic,” referring to the dispersants employedherein, is used in its conventional sense to indicate a molecularspecies having a hydrophobic region and a hydrophilic region. Thedispersants herein are “multiply amphipathic” in that they contain twoor more hydrophobic regions and two or more hydrophilic regions.

[0028] The term “attached,” as in, for example, the “attachment” of adispersant to a nanoparticle surface, includes covalent binding,adsorption, and physical immobilization. The terms “associated with,”“binding” and “bound” are identical in meaning to the term “attached.”

[0029] Attachment of the present multiply amphipathic dispersants to thesurface of a metallic or semiconductive nanoparticle will generallyinvolve “adsorption,” wherein “adsorption” refers to the noncovalentretention of a molecule by a substrate surface. That is, adsorptionoccurs as a result of noncovalent interaction between a substratesurface and adsorbing moieties present on the molecule that is adsorbed.Adsorption may occur through hydrogen bonding, van der Waal's forces,polar attraction or electrostatic forces (i.e., through ionic bonding),and in the present case will typically involve the natural affinity of ahydrophobic region of a molecule for a hydrophobic surface.

[0030] The term “nanoparticle” refers to a particle, generally asemiconductive or metallic particle, having a diameter in the range ofabout 1 nm to about 1000 nm, preferably in the range of about 2 m toabout 50 mm, more preferably in the range of about 2 nm to about 20 nm.As discussed elsewhere herein, semiconductive, and metallic“nanoparticles” generally include a passivating layer of awater-insoluble organic material that results from the method used tomanufacture such nanoparticles. The terms “surface-modifiednanoparticle” and “water-dispersible nanoparticle” as used herein referto the modified nanoparticles of the invention, while the term“nanoparticle,” without qualification, refers to the hydrophobicnanoparticle that serves as the inner core of the surface-modified,water-dispersible nanoparticle.

[0031] The terms “semiconductor nanoparticle” and “semiconductivenanoparticle” refer to a nanoparticle as defined above that is composedof an inorganic semiconductive material, an alloy or other mixture ofinorganic semiconductive materials, an organic semiconductive material,or an inorganic or organic semiconductive core contained within one ormore semiconductive overcoat layers.

[0032] The term “metallic nanoparticle” refers to a nanoparticle asdefined above that is composed of a metallic material, an alloy or othermixture of metallic materials, or a metallic core contained within oneor more metallic overcoat layers.

[0033] The terms “semiconductor nanocrystal,” “quantum dot” and “Qdot®nanocrystal” are used interchangeably herein to refer to semiconductornanoparticles composed of an inorganic crystalline material that isluminescent (i.e., they are capable of emitting electromagneticradiation upon excitation), and include an inner core of one or morefirst semiconductor materials that is optionally contained within anovercoating or “shell” of a second semiconductor material. Asemiconductor nanocrystal core surrounded by a semiconductor shell isreferred to as a “core/shell” semiconductor nanocrystal. The surroundingshell material will preferably have a bandgap energy that is larger thanthe bandgap energy of the core material and may be chosen to have anatomic spacing close to that of the core substrate. Suitablesemiconductor materials for the core and/or shell include, but are notlimited to, the following: materials comprised of a first elementselected from Groups 2 and 12 of the Periodic Table of the Elements anda second element selected from Group 16 (e.g., ZnS, ZnSe, ZnTe, CDs,CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS,SrSe, SrTe, BaS, BaSe, BaTe, and the like); materials comprised of afirst element selected from Group 13 of the Periodic Table of theElements and a second element selected from Group 15 (GaN, GaP, GaAs,GaSb, InN, InP, InAs, InSb, and the like); materials comprised of aGroup 14 element (Ge, Si, and the like); materials such as PbS, PbSe andthe like; and alloys and mixtures thereof. As used herein, all referenceto the Periodic Table of the Elements and groups thereof is to the newIUPAC system for numbering element groups, as set forth in the Handbookof Chemistry and Physics, 81^(st) 5′ Edition (CRC Press, 2000).

[0034] By “luminescence” is meant the process of emittingelectromagnetic radiation (light) from an object. Luminescence resultswhen a system undergoes a transition from an excited state to a lowerenergy state with a corresponding release of energy in the form of aphoton. These energy states can be electronic, vibrational, rotational,or any combination thereof. The transition responsible for luminescencecan be stimulated through the release of energy stored in the systemchemically or added to the system from an external source. The externalsource of energy can be of a variety of types including chemical,thermal, electrical, magnetic, electromagnetic, and physical, or anyother type of energy source capable of causing a system to be excitedinto a state higher in energy than the ground state. For example, asystem can be excited by absorbing a photon of light, by being placed inan electrical field, or through a chemical oxidation-reduction reaction.The energy of the photons emitted during luminescence can be in a rangefrom low-energy microwave radiation to high-energy x-ray radiation.Typically, luminescence refers to photons in the range from UV to IRradiation.

[0035] The term “monodisperse” refers to a population of particles(e.g., a colloidal system) wherein the particles have substantiallyidentical size and shape. For the purpose of the present invention, a“monodisperse” population of particles means that at least about 60% ofthe particles, preferably about 75% to about 90% of the particles, fallwithin a specified particle size range. A population of monodisperseparticles deviates less than 10% rms (root-mean-square) in diameter andpreferably less than 5% rms.

[0036] The phrase “one or more sizes of nanoparticles” is usedsynonymously with the phrase “one or more particle size distributions ofnanoparticles.” One of ordinary skill in the art will realize thatparticular sizes of nanoparticles such as semiconductor nanocrystals areactually obtained as particle size distributions.

[0037] By use of the term “narrow wavelength band” or “narrow spectrallinewidth” with regard to the electromagnetic radiation emission of thesemiconductor nanocrystal is meant a wavelength band of emissions notexceeding about 60 nm, and preferably not exceeding about 30 nm inwidth, more preferably not exceeding about 20 nm in width, and symmetricabout the center. It should be noted that the bandwidths referred to aredetermined from measurement of the full width of the emissions at halfpeak height (FWHM), and are appropriate in the range of 200 nm to 2000nm.

[0038] By use of the term “a broad wavelength band,” with regard to theexcitation of the semiconductor nanocrystal is meant absorption ofradiation having a wavelength equal to, or shorter than, the wavelengthof the onset radiation (the onset radiation is understood to be thelongest wavelength (lowest energy) radiation capable of being absorbedby the semiconductor nanocrystal). This onset occurs near to, but atslightly higher energy than the “narrow wavelength band” of theemission. This is in contrast to the “narrow absorption band” of dyemolecules, which occurs near the emission peak on the high energy side,but drops off rapidly away from that wavelength and is often negligibleat wavelengths further than 100 nm from the emission.

[0039] The term “emission peak” refers to the wavelength of light withinthe characteristic emission spectra exhibited by a particularsemiconductor nanocrystal size distribution that demonstrates thehighest relative intensity.

[0040] The term “excitation wavelength” refers to light having awavelength lower than the emission peak of the semiconductor nanocrystalused in the first detection reagent.

[0041] A “hydrophobic” compound (e.g., a “hydrophobic” monomer) is onethat will transfer from an aqueous phase to an organic phase,specifically from water to an organic, water-immiscible nonpolar solventwith a dielectric constant <5, with a partition coefficient of greaterthan about 50%. A “hydrophobic monomer unit” refers to a hydrophobicmonomer as it exists within a polymer. A “hydrophobic region” refers toa hydrophobic molecular segment, e.g., a molecular segment within apolymer. A “hydrophobic region” may be a single hydrophobic monomer unitor two or more hydrophobic monomer units that may be the same ordifferent and may or may not be adjacent.

[0042] A “hydrophilic” compound (e.g., a “hydrophilic” monomer) is onethat will transfer from an organic phase to an aqueous phase,specifically from an organic, water-immiscible nonpolar solvent with adielectric constant ≦5 to water, with a partition coefficient of greaterthan about 50%. A “hydrophilic monomer unit” refers to a hydrophilicmonomer as it exists in a polymeric segment or polymer. A “hydrophilicregion” refers to a hydrophilic molecular segment, e.g., a hydrophilicmolecular segment within a polymer. A “hydrophilic region” may be asingle hydrophilic monomer unit or two or more hydrophilic monomer unitsthat may be the same or different and may or may not be adjacent.

[0043] The term “ionizable” refers to a group that is electronicallyneutral at a specific pH, but can be ionized and thus renderedpositively or negatively charged at higher or lower pH, respectively.

[0044] The term “alkyl” as used herein refers to a branched orunbranched saturated hydrocarbon group of 1 to approximately 24 carbonatoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl and tetracosyl, aswell as cycloalkyl groups such as cyclopentyl and cyclohexyl. The term“lower alkyl” intends an alkyl group of 1 to 4 carbon atoms, and thusincludes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, andt-butyl.

[0045] The term “alkylene” as used herein refers to a difunctionalsaturated branched or unbranched hydrocarbon chain containing from 1 toapproximately 24 carbon atoms, typically 1 to approximately 12 carbonatoms, and includes, for example, methylene (—CH₂—), ethylene(—CH₂—CH₂—), propylene (—CH₂—CH₂—CH₂—), 2-methylpropylene(—CH₂—CH(CH₃)—CH₂—), hexylene (—(CH₂)₆—), and the like. “Loweralkylene,” as in the lower alkylene linkage of the optional couplingagent herein, refers to an alkylene group of 1 to 4 carbon atoms.

[0046] The term “alkenyl” as used herein refers to a branched orunbranched hydrocarbon group typically although not necessarilycontaining 2 to about 24 carbon atoms and at least one double bond, suchas ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, octenyl,decenyl, and the like. Generally, although not necessarily, alkenylgroups herein contain 2 to about 12 carbon atoms. The term “loweralkenyl” intends an alkenyl group of 2 to 4 carbon atoms, and the term“alkenylene” refers to a difunctional alkenyl group, in the same waythat the term “alkylene” refers to a difunctional alkyl group.

[0047] The term “alkynyl” as used herein refers to a branched orunbranched hydrocarbon group typically although not necessarilycontaining 2 to about 24 carbon atoms and at least one triple bond, suchas ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl, octynyl,decynyl, and the like. Generally, although again not necessarily,alkynyl groups herein contain 2 to about 12 carbon atoms. The term“lower alkynyl” intends an alkynyl group of 2 to 4 carbon atoms,preferably 3 or 4 carbon atoms.

[0048] The term “heteroatom-containing” and the prefix “hetero-,” as in“heteroatom-containing alkyl” and “heteroalkyl,” refer to a molecule ormolecular fragment in which one or more carbon atoms is replaced with anatom other carbon, e.g., nitrogen, oxygen, sulfur, phosphorus orsilicon.

[0049] The term “alkoxy” as used herein refers to a substituent —O—Rwherein R is alkyl as defined above. The term “lower alkoxy” refers tosuch a group wherein R is lower alkyl as defined above, e.g., methoxy,ethoxy and the like. The term “aryl” as used herein, and unlessotherwise specified, refers to an aromatic moiety containing 1 to 3aromatic rings. For aryl groups containing more than one aromatic ring,the rings may be fused or linked. Aryl groups are optionally substitutedwith one or more inert, nonhydrogen substituents per ring; suitable“inert, nonhydrogen” substituents include, for example, halo, haloalkyl(preferably halo-substituted lower alkyl), alkyl (preferably loweralkyl), alkenyl (preferably lower alkenyl), alkynyl (preferably loweralkynyl), alkoxy (preferably lower alkoxy), alkoxycarbonyl (preferablylower alkoxycarbonyl), carboxy, nitro, cyano and sulfonyl. Unlessotherwise indicated, the term “aryl” is also intended to includeheteroaromatic moieties, i.e., aromatic heterocycles. Generally,although not necessarily, the heteroatoms will be nitrogen, oxygen orsulfur. The term “arylene” refers to a difunctional aryl moiety in thesame way that the term “alkylene” refers to a difunctional alkyl group.

[0050] The term “aralkyl” refers to an alkyl group with an arylsubstituent, and the term “aralkylene” refers to an alkylene group withan aryl substituent; the term “alkaryl” refers to an aryl group that hasan alkyl substituent, and the term “alkarylene” refers to an arylenegroup with an alkyl substituent.

[0051] The terms “halo” and “halogen” are used in the conventional senseto refer to a chloro, bromo, fluoro, or iodo substituent. The term“haloalkyl” refers to an alkyl group in which at least one of thehydrogen atoms in the group has been replaced with a halogen atom.

[0052] The term “peptide” refers to oligomers or polymers of any lengthwherein the constituent monomers are alpha amino acids linked throughamide bonds, and encompasses amino acid dimers as well as polypeptides,peptide fragments, peptide analogs, naturally occurring proteins,mutated, variant, or chemically modified proteins, fusion proteins, andthe like. The amino acids of the peptide molecules may be any of thetwenty conventional amino acids, stereoisomers (e.g., D-amino acids) ofthe conventional amino acids, structural variants of the conventionalamino acids, e.g., iso-valine, or non-naturally occurring amino acidssuch as α,α-disubstituted amino acids, N-alkyl amino acids, β-alanine,naphthylalanine, 3-pyridylalanine, 4-hydroxyproline, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,and nor-leucine. In addition, the term “peptide” encompasses peptideswith posttranslational modifications such as glycosylations,acetylations, phosphorylations, and the like. The term “oligonucleotide”is used herein to include a polymeric form of nucleotides of any length,either ribonucleotides or deoxyribonucleotides. This term refers only tothe primary structure of the molecule. Thus, the term includes triple-,double- and single-stranded DNA, as well as triple-, double- andsingle-stranded RNA. It also includes modifications, such as bymethylation and/or by capping, and unmodified forms of theoligonucleotide. More particularly, the term includespolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing normucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oregon, as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers, providing that the polymerscontain nucleobases in a configuration that allows for base pairing andbase stacking, such as is found in DNA and RNA. There is no intendeddistinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms refer only to the primary structure of the molecule. Thus, theseterms include, for example, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotideN3′ P5′ phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, DNA:RNAhybrids, and hybrids between PNAs and DNA or RNA, and also include knowntypes of modifications, for example, labels which are known in the art,methylation, “caps,” substitution of one or more of the naturallyoccurring nucleotides with an analog, internucleotide modifications suchas, for, example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.),with negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), and with positively charged linkages (e.g.,aminoalklyphosphoramidates, aminoalkylphosphotriesters), thosecontaining pendant moieties, such as, for example, proteins (includingnucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide or oligonucleotide.

[0053] The term “polymer” is used herein in its conventional sense torefer to a compound having two or more monomer units, and is intended toinclude linear and branched polymers, the term “branched polymers”encompassing simple branched structures as well as hyperbranched anddendritic polymers. The term “monomer” is used herein to refer tocompounds that are not polymeric. “Polymers” herein may be naturallyoccurring, chemically modified, or chemically synthesized.

[0054] The term “water-dispersible” as used herein refers to anessentially unaggregated dispersion of particles, such that discreteparticles of approximately 2 nm to 50 nm can be sustained indefinitelyat high concentrations (10-20 μM).

[0055] The term “binding pair” refers to first and second molecules thatspecifically bind to each other. “Specific binding” of the first memberof the binding pair to the second member of the binding pair in a sampleis evidenced by the binding of the first member to the second member, orvice versa, with greater affinity and specificity than to othercomponents in the sample. The binding between the members of the bindingpair is typically noncovalent. The terms “affinity molecule” and “targetanalyte” are also used herein to refer to the first and second membersof a binding pair, respectively. Exemplary binding pairs include anyhaptenic or antigenic compound in combination with a correspondingantibody or binding portion or fragment thereof (e.g., digoxigenin andanti-digoxigenin; mouse immunoglobulin and goat anti-mouseimmunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin,biotin-streptavidin, hormone [e.g., thyroxine and cortisol]-hormonebinding protein, receptor-receptor agonist or antagonist (e.g.,acetylcholine receptor-acetylcholine or an analog thereof), IgG-proteinA, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor,and complementary polynucleotide pairs capable of forming nucleic acidduplexes), and the like.

[0056] A “nanoparticle conjugate” refers to a nanoparticle linked,through an outer layer of an amphipathic dispersant, to a member of a“binding pair” that will selectively bind to a detectable substancepresent in a sample, e.g., a biological sample. The first member of thebinding pair linked to the nanoparticle can comprise any molecule, orportion of any molecule, that is capable of being linked to thenanoparticle and that, when so linked, is capable of specificallyrecognizing the second member of the binding pair.

[0057] All molecular weights specified herein are number averagemolecular weights.

[0058] II. The nanoparticles:

[0059] Prior to surface modification with a multiply amphipathicdispersant, the nanoparticles of the invention are nanoparticles withhydrophobic surfaces, the particles having a diameter in the range ofabout 1 nm to about 1000 nm, preferably in the range of about 2 nm toabout 50 nm, more preferably in the range of about 2 nm to about 20 nm.Generally, the nanoparticles will be comprised of a semiconductive ormetallic material, with semiconductive nanoparticles preferred. Also, aswill be explained in greater detail below, the semiconductive ormetallic material typically has a coating of a hydrophobic passivatinglayer resulting from the use of solvents and/or surfactants duringnanoparticle manufacture. The hydrophobic surfaces of the nanoparticleshave affinity for and thus serve to attach the amphipathic dispersant byvirtue of the hydrophobic regions within the dispersant.

[0060] Semiconductive nanoparticles may be composed of an organicsemiconductor material or an inorganic semiconductor material. Organicsemiconductor materials will generally be conjugated polymers. Suitableconjugated polymers include, for example, cis and trans polyacetylenes,polydiacetylenes, polyparaphenylenes, polypyrroles, polythiophenes,polybithiophenes, polyisothianaphthene, polythienylvinylenes,polyphenylenesulfide, polyaniline, polyphenylenevinylenes, andpolyphenylenevinylene derivatives, e.g.,poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene vinylene (“MEH-PPV”)(see U.S. Pat. No. 5,189,136 to Wudl et al.), poly(2,5-bischelostanoxy-1,4-phenylene vinylene) (“BCHA-PPV”) (e.g., asdescribed in International Patent Publication No. WO 98/27136), andpoly(2-N,N-dimethylamino phenylene vinylene)(described in U.S. Pat. No.5,604,292 to Stenger-Smith et al.). Inorganic semiconductivenanoparticles are, however, preferred, and are optimally crystalline innature; such nanoparticles are termed “semiconductor nanocrystals”herein. Semiconductor nanocrystals are capable of luminescence,generally fluorescence, when excited by light. Currently, detection ofbiological compounds by photoluminescence utilizes fluorescent organicdyes and chemiluminescent compounds. The use of semiconductornanocrystals as luminescent markers, particularly in biological systems,provides advantages over existing fluorescent dyes. Many of theseadvantages relate to the spectral properties of nanocrystals, e.g., theability to control the composition and size of nanocrystals enables oneto construct nanocrystals with fluorescent emissions at any wavelengthin the UV-visible-IR regions. With respect to composition, for example,semiconductor nanocrystals that emit energy in the visible rangeinclude, but are not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, andGaAs. Semiconductor nanocrystals that emit energy in the near IR rangeinclude, but are not limited to, InP, InAs, InSb, PbS, and PbSe.Finally, semiconductor nanocrystals that emit energy in the blue tonear-ultraviolet include, but are not limited to, ZnS and GaN. For anyparticular nanocrystal composition, it is also possible to tune theemission to a desired wavelength by controlling particle sizedistribution. In preferred embodiments, 5-20 discrete emissions (five totwenty different size populations or distributions distinguishable fromone another), more preferably 10-15 discrete emissions, are obtained forany particular composition, although one of ordinary skill in the artwill realize that fewer than five emissions and more than twentyemissions could be obtained depending on the monodispersity of thesemiconductor nanocrystal particle population. If high informationdensity is required, and thus a greater number of distinct emissions,the nanocrystals are preferably substantially monodisperse within thesize range given above.

[0061] As explained above, “monodisperse” refers to a population ofparticles (e.g., a colloidal system) in which the particles havesubstantially identical size and shape. In preferred embodiments forhigh information density applications, monodisperse particles deviateless than 10% rms in diameter, and preferably less than 5% rms.Monodisperse semiconductor nanocrystals have been described in detail inMurray et al. (1993) J. Am. Chem. Soc. 115:8706, and in Murray,“Synthesis and Characterization of II-VI Quantum Dots and Their Assemblyinto 3-D Quantum Dot Superlattices,” doctoral dissertation,Massachusetts Institute of Technology (1995). One of ordinary skill inthe art will also realize that the number of discrete emissions that canbe distinctly observed for a given composition depends not only upon themonodispersity of the particles, but also on the deconvolutiontechniques employed. Semiconductor nanocrystals, unlike dye molecules,can be easily modeled as Gaussians and therefore are more easily andmore accurately deconvoluted.

[0062] However, for some applications, high information density will notbe required and it may be more economically attractive to use morepolydisperse particles. Thus, for applications that do not require highinformation density, the linewidth of the emission may be in the rangeof 40-60 nm.

[0063] Semiconductor nanocrystals may be made using techniques known inthe art. See, e.g., U.S. Pat. Nos. 6,048,616, 5,990,479, 5,690,807,5,505,928 and 5,262,357, as well as International Patent Publication No.WO 99/26299, published May 27, 1999. In particular, exemplary materialsfor use as semiconductor nanocrystals in the biological and chemicalassays of the present invention include, but are not limited to, thosedescribed above, including Group 2-16, 12-16, 13-15 and 14semiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe,MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs,GaSb, InP, InAs, InSb, AIS, AIP, AlSb, PbS, PbSe, Ge and Si and ternaryand quaternary mixtures thereof.

[0064] In a preferred embodiment, the surface of the semiconductornanocrystal is modified to enhance the efficiency of the emissions,prior to surface modification with the multiply amphipathic dispersant,by adding an overcoating layer or shell to the semiconductornanocrystal. The shell is preferred because at the surface of thesemiconductor nanocrystal, surface defects can result in traps forelectrons or holes that degrade the electrical and optical properties ofthe semiconductor nanocrystal. An insulating layer at the surface of thesemiconductor nanocrystal provides an atomically abrupt jump in thechemical potential at the interface that eliminates energy states thatcan serve as traps for the electrons and holes. This results in higherefficiency in the luminescent process.

[0065] Suitable materials for the shell include semiconductor materialshaving a higher bandgap energy than the semiconductor nanocrystal core.In addition to having a bandgap energy greater than the semiconductornanocrystal core, suitable materials for the shell should have goodconduction and valence band offset with respect to the coresemiconductor nanocrystal. Thus, the conduction band is desirably higherand the valence band is desirably lower than those of the coresemiconductor nanocrystal. For semiconductor nanocrystal cores that emitenergy in the visible (e.g., CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, GaAs) ornear IR (e.g., InP, InAs, InSb, PbS, PbSe), a material that has abandgap energy in the ultraviolet regions may be used. Exemplarymaterials include ZnS, GaN, and magnesium chalcogenides, e.g., MgS,MgSe, and MgTe. For a semiconductor nanocrystal core that emits in thenear IR, materials having a bandgap energy in the visible, such as CdSor CdSe, may also be used. The preparation of a coated semiconductornanocrystal may be found in, e.g., Dabbousi et al. (1997) J. Phys. Chem.B 101:9463, Hines et al. (1996) J. Phys. Chem. 100: 468-471, Peng et al.(1997) J. Am. Chem. Soc. 119:7019-7029, and Kuno et al. (1997) J. Phys.Chem. 106:9869.

[0066] The nanoparticles of the invention may also be metallic. Suchparticles are useful, for example, in surface enhanced Raman scattering(SERS), which employs nanometer-size particles onto which Raman activemoieties (e.g., a dye or pigment, or a functional group exhibiting acharacteristic Raman spectrum) are adsorbed or attached. Metallicnanoparticles may be comprised of any metal or metallic alloy orcomposite, although for use in SERS, a SERS active metal is used, e.g.,silver, gold, copper, lithium, aluminum, platinum, palladium, or thelike. In addition, the particles can be in a core-shell configuration,e.g., a gold core may be encased in a silver shell; see, e.g., Freemanet al. (1996) J. Phys. Chem. 100:718-724, or the particles may formsmall aggregates in solution. Kneipp et al. (1998) Applied Spectroscopy52:1493.

[0067] Generally, and as alluded to above, the unmodifiednanoparticles—and thus the inner core of the present surface-modifiednanoparticles—also comprise a hydrophobic coating on the semiconductiveor metallic material resulting from solvents and/or surfactants used innanoparticle manufacture. For example, semiconductive nanoparticles, asmanufactured, will typically have a water-insoluble organic coating thathas affinity for the semiconductive material, the coating comprised of apassivating layer resulting from use of a coordinating solvent such ashexyldecylamine or a trialkyl phosphine or trialkyl phosphine oxide,e.g., trioctylphosphine oxide (TOPO), trioctylphosphine (TOP), ortributylphosphine (TBP). Hydrophobic surfactants typically used in themanufacture of metallic nanoparticles and forming a coating thereoninclude, by way of example, octanethiol, dodecanethiol, dodecylamine,and tetraoctylammonium bromide. Metallic inner cores will typically havea surfactant coating that has affinity for the metallic material, thecoating similarly deriving from surfactant compounds used in themanufacture of metallic nanoparticles. The surfactant coating iscomprised of a hydrophobic surfactant.

[0068] III. The dispersant:

[0069] The dispersant used to modify the hydrophobic surface of thenanoparticles is a multiply amphipathic dispersant, i.e., a compoundhaving two or more hydrophobic regions and two or more hydrophilicregions. In a preferred embodiment, the multiply amphipathic dispersantis polymeric, and may be composed of either a linear or branchedpolymer, whether naturally occurring, chemically modified, or chemicallysynthesized. Structurally, polymers are classified as either linear orbranched wherein the term “branched” generally means that the individualmolecular units (i.e., monomer units) of the branches are discrete fromthe polymer backbone, and may or may not have the same chemicalconstitution as the polymer backbone.

[0070] As will be appreciated by those of ordinary skill in the art, thesimplest branched polymers are the “comb branched” polymers wherein alinear backbone bears one or more essentially linear pendant sidechains. This simple form of branching may be regular or irregular (inthe latter case, the branches are distributed in non-uniform or randomfashion on the polymer backbone). An example of regular comb branchingis a comb branched polystyrene as described by Altores et al. (1965) J.Polymer Sci., Part A 3:4131-4151, and an example of irregular combbranching is illustrated by the graft copolymers described by Sorensonet al. in Preparative Methods of Polymer Chemistry, 2nd Ed.,Interscience Publishers, pp. 213-214 (1968).

[0071] The amphipathic dispersant may also be a branched polymer in theform of a cross-linked or network polymer, i.e., a polymeric structurewherein individual polymer chains or branches are connected through theuse of bifunctional compounds; e.g., acrylic acid monomer units bridgedor crosslinked with a diamine linker. In this type of branching, many ofthe individual branches are not linear in that each branch may itselfcontain side chains pendant from a linear chain and it is not possibleto differentiate between the backbone and the branches. Moreimportantly, in network branching, each polymer macromolecule (backbone)is cross-linked at two or more sites to other polymer macromolecules.Also, the chemical constitution of the cross-linkages may vary from thatof the polymer macromolecules. In this cross-linked or network branchedpolymer, the various branches or cross-linkages may be structurallysimilar (termed “regularly” cross-linked) or they may be structurallydissimilar (termed “irregularly” cross-linked).

[0072] The amphipathic dispersant may also have other structuralconfigurations, e.g., it may be a star/comb-branched type polymer, asdescribed in U.S. Pat. Nos. 4,599,400 and 4,690,985, or a rod-shapeddendrimer as disclosed in U.S. Pat. No. 4,694,064.

[0073] Particularly preferred amphipathic dispersants herein arehyperbranched (containing two or more generations of branching) ordendrimeric. In contrast to hyperbranched polymers, dendrimers areregularly branched macromolecules with a branch point at each repeatunit. Also, hyperbranched polymers are obtained via a polymerizationreaction, while most regular dendrimers are obtained by a series ofstepwise coupling and activation steps. Examples of dendrimers includethe polyamidoamine (PAMAM) Starburst® dendrimers of Tomalia et al.(1985) Polym. J. 17:117, the convergent dendrimers of Hawker et al.(1990) J. Am. Chem. Soc. 112:7638, and diaminobutane dendrimers,described in Tomalia et al. (1990) Angew. Chem., Int. Ed. Engl.29:135-175. With both hyperbranched polymers and dendrimers, however,the increased number of hydrophobic and hydrophilic regions amplifiesthe effect of the dispersant on the nanoparticle core, with respect toboth affinity for the nanoparticle surface (i.e., affinity of thehydrophobic regions of the dispersant for the hydrophobic surface of thenanoparticle) and water dispersibility (as a result of the increasednumber of hydrophilic regions or segments).

[0074] The hydrophilic regions represent approximately 30 wt. % to 75wt. % of the amphipathic dispersant, and are comprised of at least onemonomer unit containing an ionizable or polar moiety, preferably anionizable moiety such as a carboxylic acid, sulfonic acid, phosphonicacid or amine substituent. Examples of hydrophilic monomer unitsinclude, but are not limited to:

[0075] water-soluble ethylenically unsaturated C₃-C₆ carboxylic acids,such as acrylic acid, alkyl acrylic acids (particularly methacrylicacid), itaconic acid, maleic acid, fumaric acid,acrylamidomethyl-propanesulfonic acid, vinyl sulfonic acid, vinylphosphonic acid, vinyllactic acid, and styrene sulfonic acid;

[0076] allylamine and allylamine salts formed with an inorganic acid,e.g., hydrochloric acid;

[0077] di-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylates and methacrylates suchdimethylaminoethyl acrylate, dimethylaminoethyl methacrylate,diethylaminoethyl acrylate, diethylaminoethyl methacrylate,dimethylaminopropyl acrylate, dimethylaminobutyl acrylate,dimethylaminoneopentyl acrylate and dimethylaminoneopentyl methacrylate;

[0078] olefinically unsaturated nitriles, such as acrylonitrile;

[0079] diolefinically unsaturated monomers, particularly diallylammoniumcompounds such as dimethyldiallylammonium chloride,dimethyldiallylammonium bromide, diethyldiallylammonium chloride,methyl-t-butyldiallylammonium methosulfate,methyl-n-propyldiallylammonium chloride, dimethyldiallylammoniumhydrogensulfate, dimethyldiallylammonium dihydrogenphosphate,di-n-butyldiallylammonium bromide, diallylpiperidinium bromide,diallylpyrrolidinium chloride and diallylmorpholinium bromide;

[0080] N-vinylpyrrolidone;

[0081] N-vinylformamide;

[0082] acrylamide and substituted acrylamides, such asN-methylolacrylamide and C₁-C₃ alkyl acrylamides, particularlymethacrylamide;

[0083] N-vinylimidazole and N-vinylimidazoline; and

[0084] other monomers, typically ethylenically unsaturated monomers,preferably vinyl monomers, substituted with at least one hydrophilicfunctionality such as a carboxylate, a thiocarboxylate, an amide, animide, a hydrazine, a sulfonate, a sulfoxide, a sulfone, a sulfite, aphosphate, a phosphonate, a phosphonium, an alcohol, a thiol, a nitrate,an amine, an ammonium, or an alkyl ammonium group —[NHR¹R²]⁺, wherein R¹and R² are alkyl substituents and the group is associated with anegatively charged anion, e.g., a halogen ion, nitrate, etc. Thehydrophilic functionality may be directly bound to a carbon atom in thepolymer backbone, but will usually be bound through a linkage thatprovides some degree of spacing between the polymer backbone and thehydrophilic functional group. Suitable linkages include, but are notlimited to, branched or unbranched alkylene, branched or unbranchedalkenylene, branched or unbranched heteroalkylene (typically alkylenecontaining one or more ether or —NH— linkages) a branched or unbranchedheteroalkenylene (again, typically alkenylene containing one or moreether or —NH— linkages), arylene, heteroarylene, alkarylene, aralkylene,and the like. The linkage will typically contain 2 to 24, more typically2 to 12, carbon atoms.

[0085] The hydrophilic regions may also be composed of partially orfully hydrolyzed poly(vinyl alcohol), poly(ethylene glycol),poly(ethylene oxide), highly hydrated poly(alkylene oxides) such aspoly(ethylene oxide), cellulosic segments (e.g., comprised of celluloseper se or cellulose derivatives such as hydroxypropyl cellulose,hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, ethyl cellulose, cellulose acetate, and the like), andpolysaccharides such as chitosan or dextran.

[0086] The hydrophobic regions represent approximately 25 wt. % to 90wt. % of the amphipathic dispersant, and are comprised of at least onenon-ionizable, nonpolar monomer unit, facilitating noncovalentassociation with the hydrophobic surface of the nanoparticle. Examplesof such monomer units include, but are not limited to:

[0087] acrylates such as methacrylate, methyl methacrylate, ethylmethacrylate, butyl methacrylate, isobutyl methacrylate, hexylmethacrylate, isodecyl methacrylate, lauryl methacrylate, phenylmethacrylate, isopropyl acrylate, isobutyl acrylate andoctadecylacrylate,

[0088] alkylenes such as ethylene and propylene;

[0089] C₄-C₁₂-alkyl-substituted ethyleneimine;

[0090] alkyl acrylamides wherein the alkyl group is larger than loweralkyl (particularly alkyl acrylamides wherein the alkyl group has six ormore carbon atoms, typically six to twelve carbon atoms, such ashexylacrylamide, octylacrylamide, and the like);

[0091] styrene and hydrophobically derivatized styrenes (i.e., styrenesubstituted with one or more hydrophobic substituents, e.g., C₅-C₁₂hydrocarbyl groups);

[0092] vinyl ether;

[0093] vinyl esters such as vinyl acetate; and

[0094] vinyl halides such as vinyl chloride.

[0095] The hydrophobic regions may also be composed of polychloroprene,polybutadiene, polysiloxane, polydimethylsiloxane, polyisobutylene orpolyurethane blocks, or they may be polycondensates of2-poly(hydroxyalkanoic acids) such as 2-hydroxypropanoic acid,2-hydroxybutanoic acid, 2-hydroxyisobutanoic acid, 2-hydroxyheptanoicacid, 10-hydroxydecanoic acid, 12-hydroxydodecanoic acid,12-hydroxystearic acid, 16-hydroxyhexadecanoic acid, 2-hydroxystearicacid, 2-hydroxyvaleric acid or the corresponding condensates obtainedfrom lactones, condensates of diols and dicarboxylic acids such aspolyethylene adipate, or polylactams such as polycaprolactam.

[0096] Any of the aforementioned monomer units and polymer segments canbe modified using techniques and reagents routinely used by those ofordinary skill in the art. Such modifications include, for example,routine substitutions, additions of chemical groups such as alkyl groupsand alkylene groups, hydroxylations, oxidations, and the like. Suchbranched polymers, composed of hydrophobic segments and hydrophilicsegments, are typically comprised of (1) a hydrophobic backbone withhydrophilic branches, (2) a hydrophilic backbone with hydrophobicbranches, or (3) a backbone that may be either hydrophobic orhydrophilic, and is substituted with both hydrophilic and hydrophobicbranches. Such polymers can be prepared by any suitable method readilyknown to those of ordinary skill in the art and/or described in thepertinent texts and literature. Polymers of type (1), for example, canbe prepared by copolymerization of a hydrophobic monomer with a secondmonomer that includes suitable reactive groups through which thehydrophilic side chains (branches) can be grafted to the backbone.Alternatively, type (1) polymers can be prepared by polymerizing asingle hydrophobic monomer with a suitable reactive side group, and afraction of those reactive side groups can be modifiedpost-polymerization by grafting hydrophilic side chains. Analogously,polymers of type (2) can be prepared by copolymerization of ahydrophilic monomer with a second monomer that includes suitablereactive groups through which the hydrophobic side chains (branches) canbe grafted to the backbone. Alternatively, type (2) polymers can beprepared by polymerizing a single hydrophilic monomer with a suitablereactive side group, and a fraction of those reactive side groups can bemodified post-polymerization by grafting hydrophobic side chains. Type(3) polymers can be prepared by first synthesizing a linear polymerhaving reactive sites throughout the backbone, and then graftinghydrophilic and hydrophobic side chains onto the backbone in a fashionthat may or may not be ordered.

[0097] Particularly preferred amphipathic dispersants include acrylicacid and methacrylic acid polymers modified to include hydrophobicregions, as well as copolymers of acrylic acid and/or methacrylic acidwith hydrophobic comonomers such as alkyl acrylamides. Examples of suchpolymers are poly(acrylic acid-co-octylacrylamide), poly(acrylicacid-co-hexylacrylamide), poly(methacrylic acid-co-octylacrylamide), andpoly(methacrylic acid-co-hexylacrylamide), with poly(acrylicacid-co-octylacrylamide) most preferred. The specific methodology usedto synthesize polymers suitable as the multiply amphipathic dispersantwill depend on the particular monomer types that are employed. As willbe appreciated by those of ordinary skill in the art, suitablepolymerization techniques include step polymerization, radical chainpolymerization, emulsion polymerization, ionic chain polymerization,chain copolymerization, ring-opening polymerization, livingpolymerization, polycondensation reactions, and graft polymerization. Ina preferred embodiment, the amphipathic dispersant is formed by additionpolymerization of ethylenically unsaturated monomers. Suchpolymerization reactions are generally catalyzed using metalliccatalysts (e.g., transition metal-based metallocenes, Ziegler-Nattacatalysts, Brookhart-type catalysts, etc.) and typically involvecontacting the monomer(s), catalyst, and a catalyst activator (e.g.,methyl aluminoxane, or “MAO”) at a suitable temperature at reduced,elevated or atmospheric pressure, under an inert atmosphere, for a timeeffective to produce the desired polymer. An added solvent may, ifdesired, be employed, or the monomeric compounds may serve as solvent.The reaction may be conducted under solution or slurry conditions, in asuspension, or in the gas phase. As alluded to above, branched polymerscan be prepared using this technique by introducing reactive sites intothe polymer backbone during polymerization (e.g., by incorporating somefraction of monomer units having a pendant reactive site), followed bysynthesis or grafting of branches at the reactive sites.

[0098] In a preferred embodiment, the amphipathic dispersant iscomprised of a hydrophilic backbone that has been modified to containhydrophobic anchoring groups, i.e., hydrophobic side chains that serveto “anchor” the dispersant to the nanoparticle surface. For example,hydrophilic polymers containing pendant carboxylic acid groups (e.g., asin poly(acrylic acid), [—(CH₂CH(CO₂H)]_(n)) can be readily modified tocontain a controlled number of branched or unbranched hydrophobic sidechains using methods known in the art. In one such method, the pendantcarboxylic acid groups of poly(acrylic acid) can be activated with asuitable activating agent, e.g., thionyl chloride or a carbodiimide,followed by reaction with a long chain alkylamine, e.g., a C₄-C₁₂alkylamine such as octylamine, and finally with a hydrolyzing agent suchas water. Depending on the relative quantities of the alkylamine and thehydrolyzing agent, the resulting polymer is an amphipathic polymer witha hydrophilic backbone (by virtue of the carboxylic acid groups presentafter partial hydrolysis) and hydrophobic side chains (the long chainalkyl group attached to the backbone through an amide linkage).

[0099] Within the aforementioned group of hydrophobically modifiedhydrophilic polymers are hydrophobically modified peptides, preferablyhydrophobically modified synthetic polypeptides. The use of syntheticpolypeptides allows for control over a number of factors, including themonodispersity of the molecular weight of the hydrophilic backbone, thenumber and position of modifiable groups on the backbone, and theregularity of the modification, i.e., whether the hydrophobic groups arerandomly distributed throughout the polypeptide chain or present in anordered, “regular” fashion.

[0100] Suitable polypeptides are triblock (A-B-A) copolymers, forexample, triblock copolymers of aspartate and norleucine, in which casepolynorleucine is preferably the central block “B.” Such a triblockcopolymer provides a region rich in hydrophobic side chains. In onealternative, the central block “B” can comprise a hydrophilic aminoacid, for example, poly(lysine), which can be modified via standardchemistries to include hydrophobic side chains. The carboxylate-richaspartate side chains (A) provide the polar, ionic groups that not onlyaid in rendering the nanocrystal water dispersible, but provide reactivesites or functionalizable moieties for further chemistry, such asconjugation to affinity molecules.

[0101] The polypeptide compositions of the present invention may also bemonofunctional in nature, e.g., polylysine or polyaspartate, diblockcopolymers (A-B) or triblock copolymers of three different amino acids(A-B-C). These compositions are also not restricted to lysine oraspartate, but may make use of any number of combinations of the knownamino acids. Generally, the hydrophobic regions of a polypeptide arecomprised of at least one hydrophobic amino acid and the hydrophilicregions are comprised of at least one hydrophilic amino acid. As will beappreciated by those of ordinary skill in the art, hydrophobic aminoacids include, for example, alanine, glycine, valine, leucine,isoleucine, norleucine, proline, phenylalanine, methionine, tryptophane,cysteine, and includes hydrophilic amino acids modified to includehydrophobic side chains, while hydrophilic amino acids include asparticacid, glutamic acid, lysine, arginine, histidine, asparagine, glutamine,serine, threonine and tyrosine.

[0102] The amphipathic dispersant generally although not necessarily hasa molecular weight in the range of approximately 500 to 50,000,preferably in the range of approximately 1000 to 10,000, more preferablyin the range of approximately 1000 to 5000. The dispersant may bemodified so as to contain functionalizable sites useful for covalent ornoncovalent attachment to an external molecular moiety. Thefunctionalizable sites may be present in addition to the ionizablegroups discussed above, or the ionizable groups may themselves serve asfunctionalizable sites suitable for binding an external molecularmoiety. Functionalizable sites include, for example, any of theconventional functional groups that are modified using simple,conventional chemical techniques, e.g., amino groups, nitriles,carboxylic acids, esters, acid chlorides, and the like. Preferably,although not necessarily, the functionalizable sites are spaced apartfrom the dispersant structure by an inert linking moiety, e.g., analkylene or oxyalkylene chain, typically composed of about 2 to 20carbon atoms, preferably about 4 to 10 carbon atoms, or other linkingmoieties such as those described above with respect to the spacerlinkages that may be present linking hydrophilic functional groups tothe polymer backbone.

[0103] IV. Preparation of the Surface-Modified Nanoparticles:

[0104] Hydrophobic nanoparticles may be rendered water dispersible bysurface modification with the amphipathic dispersant. That is, thehydrophobic regions of the dispersant associate with the hydrophobicnanoparticle surface, and the hydrophilic regions are externally facingand provide water dispersibility. Surface modification of thenanoparticles is carried out as follows.

[0105] Initially, a solution of the amphipathic dispersant is preparedby admixing the selected amphipathic dispersant with a suitablenonaqueous solvent, preferably a nonpolar, water-immiscible solvent suchas n-hexane or chloroform. Ionizable groups on the dispersant, ifpresent, are then converted to salt form by treatment with anappropriate acid or base, which serves as an ionizing agent. Forionizable acidic groups, suitable bases are generally inorganic bases,e.g., ammonium hydroxides or hydroxides of alkali metals (e.g., sodiumor potassium) or alkaline earth metals (e.g., magnesium or calcium). Thehydrophobic nanoparticles are dispersed in the same solvent, eitherbefore or after the aforementioned ionization step. Typically, however,the nanoparticles are added after ionization, preferably dropwise, to astirring solution of the ionized dispersant. Alternatively, thenanoparticles may be dispersed in the solvent at the outset, and thedispersant added thereto. As another alternative, two separate solutionsmay be prepared and mixed, with one solution containing the dispersantand the other solution containing the nanoparticles, with both solutionspreferably containing the same solvent. In all cases, after preparationof the nanoparticle-dispersant-solvent admixture, the admixture ispreferably stirred for several minutes to ensure complete mixing of thecomponents.

[0106] In the next step of the process, the admixture of nanoparticles,dispersant and solvent is subjected to conditions effective to result inabsorption of the dispersant by the nanoparticles. For example, theadmixture may be heated or placed under vacuum to remove the solvent,such a drying process resulting in dispersant-coated nanoparticles.Alternatively, the conditions may involve changing the polarity of thesolvent and/or changing the ionic state of the polymer.

[0107] Next, the dispersant-coated nanoparticles are transferred to anaqueous medium such as water, using solvent exchange (if thedispersant-coated nanoparticles are not previously dried) or addition ofwater or an aqueous buffer (if the dispersant-coated nanoparticles arepreviously dried). The aqueous buffer, if one is used, should beeffective to facilitate dispersion of the nanoparticles in the aqueousmedium. The water dispersion is then filtered to remove any largemicellar structures formed by excess dispersant in solution that is notassociated with the particles. These materials may then be used in anyapplications requiring aqueous-based sols of nanocrystals. Prior tousing these particles one may further increase the stability of theamphipathic coating by chemically crosslinking the individual polymerchains of the dispersant coating such that each polymer has a potentialmultiplicity of chemical bonds to other polymer chains on the particle.One of ordinary skill in the art would recognize that the crosslinkerused may be tailored to match the properties of the dispersant coating.For example, a diamine could be used to crosslink a dispersant coatingcontaining carboxylic acids. Of particular utility are crosslinkers thatcarry charges or other groups capable of stabilizing the dispersedcolloids as described herein. A diamino carboxylate or sulfonate and adiamino polyethylene glycol crosslinkers are especially useful. Asimilar chemistry would apply for crosslinkers having multiple aminemoieties, such as dendrimers, modified dendrimers, and the like.

[0108] The amount of amphipathic dispersant per unit mass of the “innercore” (i.e., per unit mass of the original, unmodified nanoparticle) inthe resulting dispersant-coated nanoparticles is proportional to thesize and surface area of the nanoparticles. Generally, the number ratioof the dispersant to the inner core will be in the range ofapproximately 50:1 to approximately 5000:1. The ratio will be closer to50:1 for smaller nanoparticles, i.e., nanoparticles less than about 5 nmin diameter (e.g., green CdSe quantum dots), and will be closer to5000:1 for larger nanoparticles, i.e., nanoparticles about 5 nm to 10 nmin diameter (e.g., red CdSe quantum dots).

[0109] V. Nanoparticle Conjugates and Associated Compositions:

[0110] The invention additionally relates to conjugates of the presentsurface-modified semiconductive nanoparticles and compositionscomprising those conjugates in association with a target analyte.

[0111] That is, the surface-modified semiconductive nanoparticles of theinvention may be conjugated to an affinity molecule that serves as thefirst member of a binding pair. Generally, although not necessarily, itis the amphipathic dispersant on the nanoparticle surface that providesthe means for linkage to the affinity molecule. As noted previously,ionizable groups present within the hydrophilic regions of theamphipathic dispersant may provide the means for linkage to the affinitymolecule, and/or other functional groups present within or introducedinto the dispersant molecule may provide the means for linkage to theaffinity molecule. The linkage will generally be covalent, and suitablelinkers are discussed in Section III, above. Suitable methods ofconjugating molecules and molecular segments to affinity molecules aredescribed, for example, in Hermanson, Bioconjugate Techniques (AcademicPress, NY, 1996).

[0112] Such semiconductive nanoparticle “conjugates,” by virtue of theaffinity molecule, can be used to detect the presence and/or quantity ofbiological and chemical compounds, interactions in biological systems,biological processes, alterations in biological processes, oralterations in the structure of biological compounds. That is, theaffinity molecule, when linked to the semiconductive nanoparticle, caninteract with a biological target that serves as the second member ofthe binding pair, in order to detect biological processes or reactions,or to alter biological molecules or processes. Preferably, theinteraction of the affinity molecule and the biological target involvesspecific binding, and can involve covalent, noncovalent, hydrophobic,hydrophilic, electrostatic, van der Waal's, or magnetic interaction.Preferably, the affinity molecule physically interacts with thebiological target.

[0113] The affinity molecule associated with the semiconductivenanoparticles can be naturally occurring or chemically synthesized, andcan be selected to have a desired physical, chemical, or biologicalproperty. Such properties include, but are not limited to, covalent andnoncovalent association with proteins, nucleic acids, signalingmolecules, prokaryotic or eukaryotic cells, viruses, subcellularorganelles and any other biological compounds. Other properties of suchmolecules include, but are not limited to, the ability to affect abiological process (e.g. cell cycle, blood coagulation, cell death,transcription, translation, signal transduction, DNA damage or cleavage,production of radicals, scavenging radicals, etc.), and the ability toalter the structure of a biological compound (e.g. crosslinking,proteolytic cleavage, radical damage, etc

[0114] In a preferred embodiment, the nanoparticle conjugate iscomprised of a semiconductive nanoparticle that emits light at a tunablewavelength and is associated with a nucleic acid. The association can bedirect or indirect. The nucleic acid can be any ribonucleic acid,deoxyribonucleic acid, dideoxyribonucleic acid, or any derivatives andcombinations thereof. The nucleic acid can also be oligonucleotides ofany length. The oligonucleotides can be single-stranded,double-stranded, triple-stranded or higher order configurations (e.g.Holliday junctions, circular single-stranded DNA, circulardouble-stranded DNA, DNA cubes, (see Seeman (1998) Ann. Rev. Biophys.Biomol. Struct. 27:225248). Among the preferred uses of the presentcompositions and methods are detecting and/or quantitating nucleic acidsas follows: (a) viral nucleic acids; (b) bacterial nucleic acids; and(c) numerous human sequences of interest, e.g. single nucleotidepolymorphisms. Without limiting the scope of the present invention,nanoparticle conjugates can comprise nanocrystals associated withindividual nucleotides, deoxynucleotides, dideoxynucleotides or anyderivatives and combinations thereof and used in DNA polymerizationreactions such as DNA sequencing, reverse transcription of RNA into DNA,and polymerase chain reactions (PCR). Nucleotides also includemonophosphate, diphosphate and triphosphates and cyclic derivatives suchas cyclic adenine monophosphate (cAMP). Other uses of nanoparticlesconjugated to nucleic acids included fluorescence in situ hybridization(FISH). In this preferred embodiment, nanocrystals are conjugated tooligonucleotides designed to hybridize to a specific sequence in vivo.Upon hybridization, the fluorescent nanocrystal tags are used tovisualize the location of the desired DNA sequence in a cell. Forexample, the cellular location of a gene whose DNA sequence is partiallyor completely known can be determined using FISH. Any DNA or RNA whosesequence is partially or completely known can be visually targeted usingFISH. For example without limiting the scope of the present invention,messenger RNA (mRNA), DNA telomeres, other highly repeated DNAsequences, and other non-coding DNA sequencing can be targeted by FISH.

[0115] The nanoparticle conjugate may also comprise a surface-modifiedsemiconductive nanoparticle as provided herein in association with amolecule or reagent for detection of biological compounds such asenzymes, enzyme substrates, enzyme inhibitors, cellular organelles,lipids, phospholipids, fatty acids, sterols, cell membranes, moleculesinvolved in signal transduction, receptors and ion channels. Theconjugate also can be used to detect cell morphology and fluid flow;cell viability, proliferation and function; endocytosis and exocytosis(Betz et al. (1996) Curr. Opin. Neurobiol. 6(3):365-71); and reactiveoxygen species (e.g., superoxide, nitric oxide, hydroxyl radicals,oxygen radicals). In addition, the conjugate can be used to detecthydrophobic or hydrophilic regions of biological systems.

[0116] Conjugates of semiconductive nanocrystals also find utility innumerous other biological and non-biological applications whereluminescent markers, particularly fluorescent markers, are typicallyused. See, for example, Haugland, R. P. Handbook of Fluorescent Probesand Research Chemicals (Molecular Probes, Eugene, Oreg. Sixth Ed. 1996;Website, www.probes.com.). Examples of areas in which the luminescentnanoparticle conjugates of the invention are useful include, withoutlimitation, fluorescence immunocytochemistry, fluorescence microscopy,DNA sequence analysis, fluorescence in situ hybridization (FISH),fluorescence resonance energy transfer (FRET), flow cytometry(Fluorescence Activated Cell Sorter; FACS) and diagnostic assays forbiological systems. For further discussion concerning the utility ofnanocrystal conjugates in the aforementioned areas, see InternationalPatent Publication No. WO 00/17642 to Bawendi et al.

[0117] It is to be understood that while the invention has beendescribed in conjunction with the preferred specific embodimentsthereof, that the foregoing description as well as the examples thatfollow are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

[0118] The following examples are intended to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the novel compositions of the invention. Efforts have beenmade to ensure accuracy with respect to numbers used (e.g., amounts,temperatures, etc), but some experimental error and deviation should, ofcourse, be allowed for. Unless indicated otherwise, parts are parts byweight, temperatures are in degrees centigrade, and pressure is at ornear atmospheric.

[0119] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of synthetic organicchemistry, biochemistry, molecular biology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); Oligonucleotide Synthesis(M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Haines & SJ.Higgins, eds., 1984); Methods in Enzymology (Academic Press, Inc.);Kirk-Othmer's Encyclopedia of Chemical Technology; and House's ModemSynthetic Reactions. All patents, patent applications, patentpublications, journal articles and other references cited herein areincorporated by reference in their entireties.

EXAMPLE 1

[0120] Synthesis of Hydrophobically Modified Hydrophilic Polymers:

[0121] A modified polyacrylic acid was prepared by diluting 100 g [0.48mol COONa] of poly(acrylic acid, sodium salt) (obtained from Aldrich,molecular weight 1200) was diluted two-fold in water and acidified in a1.0 L round bottom flask with 150 ml (1.9 mol) of concentrated HCl. Theacidified polymer solution was concentrated to dryness on a rotaryevaporator (100 mbar, 80° C.). The dry polymer was evacuated for 12hours at <10 mbar to ensure water removal. A stirbar and 47.0 g (0.24mol) of 1-[3-(dimethyl-amino)-propyl]-ethylcarbodiimide hydrochloride(EDC-Aldrich 98%) were added to the flask, then the flask was sealed andpurged with N₂, and fit with a balloon. 500 ml of anhydrousN-N,dimethylformamide (Aldrich) was transferred under positive pressurethrough a cannula to this mixture; and the flask was swirled gently todissolve the solids. 32 ml (0.19 mol) of octylamine was transferreddropwise under positive pressure through a cannula from a sealedoven-dried graduated cylinder into the stirring polymer/EDC solution,and the stirring continued for 12 hours. This solution was concentratedto <100 ml on a rotary evaporator (30 mbar, 80° C.), and the polymer wasprecipitated by addition of 200 ml di-H₂O to the cooled concentrate,which produced a gummy white material. This material was separated fromthe supernatant and triturated with 100 ml di-H₂O three more times. Theproduct was dissolved into 400 ml ethyl acetate (Aldrich) with gentleheating, and basified with 200 ml di-H₂O and 100 gN-N-N-N-tetramethylammonium hydroxide pentahydrate (0.55 mo) for 12hours. The aqueous layer was removed and precipitated to a gummy whiteproduct with 400 ml of 1.27 M HCl. The product was decanted andtriturated with 100 ml of di-H₂O twice more, after which the aqueouswashings were back-extracted into 6×100 ml portions of ethyl acetate.These ethyl acetate solutions were added to the product flask, andconcentrated to dryness (100 mbar, 60° C.). The crude polymer wasdissolved in 300 ml of methanol and purified in two aliquots over LH-20(Amersham-Pharmacia-5.5 cm×60 cm column) at a 3 ml/minute flow rate.Fractions were tested by NMR for purity, and the pure fractions werepooled, while the impure fractions were re-purified on the LH-20 column.After pooling all of the pure fractions, the polymer solution wasconcentrated by rotary evaporation to dryness, and evacuated for 12hours at <10 mbar. The product was a white powder (25.5 g, 45% oftheoretical yield), which showed broad NMR peaks in CD₃OD [δ=3.1 b(9.4), 2.3 b (9.7), 1.9 1.7 1.5 1.3 b (63.3) 0.9 bt (11.3)], and clearIR signal for both carboxylic acid (1712 cm⁻¹) and amide groups (1626cm⁻¹, 1544 cm⁻¹).

EXAMPLE 2

[0122] Preparation of Surface-Modified Nanocrystals:

[0123] Twenty milliliters of 3-5 μM (3-5 nmoles) of TOPO/TOP coatedCdSe/ZnS nanocrystals (see, Murray et al. (1993) J. Am. Chem. Soc.115:8706) were precipitated with 20 milliliters of methanol. Theflocculate was centrifuged at 3000×g for 3 minutes to form a pellet ofthe nanocrystals. The supernatant was thereafter removed and 20milliliters of methanol was again added to the particles. The particleswere vortexed to loosely disperse the flocculate throughout themethanol. The flocculate was centrifuged an additional time to form apellet of the nanocrystals. This precipitation/centrifugation step wasrepeated an additional time to remove any excess reactants remainingfrom the nanocrystal synthesis. Twenty milliliters of chloroform wereadded to the nanocrystal pellet to yield a freely dispersed sol.

[0124] 300 milligrams of hydrophobically modified poly(acrylic acid) wasdissolved in 20 ml of chloroform. Tetrabutylammonium hydroxide (1.0 M inmethanol) was added to the polymer solution to raise the solution to pH10 (pH was measured by spotting a small aliquot of the chloroformsolution on pH paper, evaporating the solvent and thereafter wetting thepH paper with distilled water). Thereafter the polymer solution wasadded to 20 ml of chloroform in a 250 ml round bottom flask equippedwith a stir bar. The solution was stirred for 1 minute to ensurecomplete admixture of the polymer solution. With continued stirring thewashed nanocrystal dispersion described above was added dropwise to thepolymer solution. The dispersion was then stirred for two minutes toensure complete mixing of the components and thereafter the chloroformwas removed in vacuo with low heat to yield a thin film of theparticle-polymer complex on the wall of the flask. Twenty milliliters ofdistilled water were added to the flask and swirled along the walls ofthe flask to aid in dispersing the particles in the aqueous medium. Thedispersion was then allowed to stir overnight at room temperature. Atthis point the nanocrystals are freely dispersed in the aqueous medium,possess pendant chemical functionalities and may therefore be linked toaffinity molecules of interest using methods well known in the art forbiolabeling experiments. In addition, the fact that the nanocrystals nowhave a highly charged surface means they can be readily utilized inpolyelectrolyte layering experiments for the formation of thin films andcomposite materials.

EXAMPLE 3

[0125] Preparation of Nanocrystal Conjugates:

[0126] Functional and specific biological labels have been made withmaterials of the present invention as follows: The polymer-stabilizedparticles from Example 1 were purified away from excess (non-absorbed)polymer and tetrabutylammonium hydroxide via tangential flowdiafiltration using a 100 K polyethersulfone membrane against one literof distilled water and one liter of 50 mM morpholinoethanesulfonic acidbuffer, pH 5.9. The purified dispersion was concentrated to 20milliliters and 10 milliliters of this nanocrystal dispersion wereactivated with 79 pmoles (15 mg) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 158 pmoles (34 mg)N-hydroxysulfosuccinimide for 30 minutes at room temperature. Theparticle dispersion was then buffer exchanged to pH 8.0 viadiafiltration against 50 mM phosphate buffer, pH 8.0. When the particledispersion reached pH 8.0, streptavidin was added to the particles at a5:1 protein:particle ratio (175 nmoles, 10.5 mg) and the reactionmixture was incubated overnight at room temperature with stirring. Afterovernight incubation the conjugated particles were separated fromexcess, unreacted protein via tangential flow diafiltration using a100,000 MW polyethersulfone membrane against 2 liters of phosphatebuffer, 50 mM, pH 7.0.

[0127] At this point these materials can be stored in any number ofbiological buffers and used as fluorescent biological labels to detectbiotin-labeled analytes of interest. Although streptavidin was used hereas an example, the simplicity and generality of the above couplingchemistries can be efficiently extended to forming functional conjugateswith any number of biological molecules of interest, such as antibodies,peptides, and oligonucleotides, for example.

EXAMPLE 4

[0128] Crosslinking of Polymer Stabilized Nanocrystals with a Dendrimer:

[0129] Ten milliliters of nanocrystals at 3.5 μM, stabilized asdescribed in Example 2, were purified by tangential flow filtration, asdescribed in Example 3, against 1 liter of distilled water to removeexcess polymer. The nanocrystals were concentrated to 10 milliliters andthe pH of the aqueous dispersion was decreased to pH 6.5 with 50 μladditions of 0.1 M HCl. 67 milligrams (315 lmoles) EDC were added to thestirring nanocrystal dispersion. The reaction was allowed to proceed for10 minutes before 1 milliliter of 0.5M borate buffer (pH 8.5) containing3.94 μmoles of the crosslinking reagent Starburst® (PAMAM) Dendrimer,Generation 0, were added to the reaction mixture. The reaction mixturewas stirred for 2 hours at room temperature and then transferred to a50,000 molecular weight cut-off polyethersulfone dialysis bag. Dialysiswas performed for 24 hours against 2 changes of 4 liters of water.

EXAMPLE 5

[0130] Crosslinking of Polymer Stabilized Nanocrystals with a DiaminoCrosslinker:

[0131] Ten milliliters of nanocrystals at 3.5 μM, stabilized asdescribed in Example 2, were purified by tangential flow filtration, asdescribed in Example 3, against 1 liter of distilled water to removeexcess polymer. The nanocrystals were concentrated to 10 milliliters andthe pH of the aqueous dispersion was decreased to pH 6.5 with 50 μladditions of 0.1M HCl. 67 milligrams (315 μmoles) EDC were added to thestirring nanocrystal dispersion. The reaction was allowed to proceed for10 minutes before 1 milliliter of 0.5M borate buffer (pH 8.5) containing3.94 μmoles of the crosslinking reagent lysine (a diamino carboxylicacid) were added to the reaction mixture. The reaction mixture wasstirred for 2 hours at room temperature and then transferred to a 50,000molecular weight cut-off polyethersulfone dialysis bag. Dialysis wasperformed for 24 hours against 2 changes of 4 liters of water.

EXAMPLE 6

[0132] Preparation of Surface Modified Nanocrystals with Polypeptides:

[0133] A triblock polypeptide comprised of(Aspartate)₄-(Norleucine)₈-(Aspartate)₄ has been used to stabilizehydrophobic nanocrystals in water by the following method: Fivemilliliters of a 3.5 μM nanocrystal solution were washed as described inExample 1 and redispersed in 5 milliliters of chloroform. 75 milligramsof an (Aspartate)₄-(Norleucine)₈-(Aspartate)₄ triblock polypeptide weredissolved in 5 milliliters of a 50:50 mixture of chloroform:methanol andthe pH of the polypeptide solution was raised to 10 with aliquots oftetrabutylammonium hydroxide (11.0M in methanol). This polypeptidesolution was then added to 5 milliliters of chloroform in a 50milliliter round bottom flask. The solution was allowed to stir for 1minute to ensure complete mixing. The washed nanocrystal dispersion inchloroform was then added dropwise to the stirring polypeptide solutionand the entire mixture was allowed to stir for an additional 2 minutesbefore all the solvent was removed in vacuo with low heat (40 degreesCelsius) to yield a thin film of the particle-polymer complex on thewall of the flask. Five milliliters of distilled water were then addedto the flask and swirled in order to aid in dispersing the nanocrystalsfully in the aqueous medium. As with the nanocrystals stabilized inExample 1, these polypeptide stabilized nanocrystals can be efficientlypurified away from excess polypeptide by dialysis, tangential flowfiltration, or various forms of chromatography known to those skilled inthe art.

We claim:
 1. A water-dispersible nanoparticle comprising: (a) an innercore comprised of a semiconductive or metallic material; (b) awater-insoluble organic coating provided thereon; and (c) an outer layerof a multiply amphipathic polymer that comprises at least twohydrophobic regions and at least two hydrophilic regions; and (d) afunctional group covalently bound to the polymer through an oxyalkylenelinking moiety.
 2. The water-dispersible nanoparticle of claim 1,wherein the oxyalkylene linking moiety is composed of 2 to about 20carbon atoms.
 3. The water-dispersible nanoparticle of claim 1, whereinthe functional group is selected from amino, cyano, carboxyl, ester, andhalocarbonyl groups.
 4. The water-dispersible nanoparticle of claim 3,wherein the functional group is an amino group.
 5. The water-dispersiblenanoparticle of claim 1, wherein the inner core is comprised of asemiconductive material.
 6. The water-dispersible nanoparticle of claim5, wherein the semiconductive material is inorganic.
 7. Thewater-dispersible nanoparticle of claim 6, wherein the semiconductivematerial is crystalline.
 8. The water-dispersible nanoparticle of claim5, wherein the water-insoluble organic coating is comprised oftrioctylphosphine oxide, trioctylphosphine, tributylphosphine, or amixture thereof.
 9. The water-dispersible nanoparticle of claim 5,further including a shell layer between the core and the water-insolubleorganic coating.
 10. The water-dispersible nanoparticle of claim 9,wherein the shell layer is comprised of a semiconductive material havinga band gap energy greater than that of the inner core.
 11. Thewater-dispersible nanoparticle of claim 1, wherein the inner core iscomprised of a metallic material.
 12. The water-dispersible nanoparticleof claim 11, wherein the water-insoluble organic coating has an affinityfor the metallic material.
 13. The water-dispersible nanoparticle ofclaim 12, wherein the water-insoluble organic coating is comprised of ahydrophobic surfactant.
 14. The water-dispersible nanoparticle of claim13, wherein the hydrophobic surfactant is selected from the groupconsisting of octanethiol, dodecanethiol, dodecylamine,tetraoctylammonium bromide, and mixtures thereof.
 15. Thewater-dispersible nanoparticle of claim 1, wherein the multiplyamphipathic polymer is linear or branched.
 16. The water-dispersiblenanoparticle of claim 15, wherein the multiply amphipathic polymer isbranched.
 17. The water-dispersible nanoparticle of claim 16, whereinthe multiply amphipathic polymer is hyperbranched or dendritic.
 18. Thewater-dispersible nanoparticle of claim 1, wherein the hydrophobicregions of the multiply amphipathic polymer are each comprised of atleast one non-ionizable, nonpolar monomer unit.
 19. Thewater-dispersible nanoparticle of claim 1, wherein each of thehydrophobic regions comprise at least one monomer unit selected from thegroup consisting of ethylene, propylene, alkyl (C₄-C₁₂)-substitutedethyleneimine, an alkyl acrylate, a methacrylate, a phenyl acrylate, analkyl acrylamide, a styrene, a hydrophobically derivatized styrene, avinyl ether, a vinyl ester, a vinyl halide, and a combination thereof.20. The water-dispersible nanoparticle of claim 19, wherein thehydrophobic regions comprise at least one monomer unit selected from analkyl acrylate, an alkyl methacrylate, an alkyl acrylamide, and amixture thereof.
 21. The water-dispersible nanoparticle of claim 1,wherein the hydrophilic regions of the multiply amphipathic polymer eachcomprise at least one monomer unit containing an ionizable or polarmoiety.
 22. The water-dispersible nanoparticle of claim 21, wherein thehydrophilic regions each comprise at least one monomer unit containingan ionizable moiety.
 23. The water-dispersible nanoparticle of claim 22,wherein the ionizable moiety is selected from the group consisting ofcarboxylic acid, sulfonic acid, phosphonic acid, and amine substituents.24. The water-dispersible nanoparticle of claim 18, wherein thehydrophilic regions each comprise at least one monomer unit containingan ionizable or polar moiety.
 25. The water-dispersible nanoparticle ofclaim 24, wherein the hydrophilic regions each comprise at least onemonomer unit containing an ionizable moiety.
 26. The water-dispersiblenanoparticle of claim 25, wherein the ionizable moiety is selected fromthe group consisting of carboxylic acid, sulfonic acid, phosphonic acid,and an amine substituent.
 27. The water-dispersible nanoparticle ofclaim 1, wherein the hydrophilic regions of the multiply amphipathicpolymer t each comprise at least one monomer unit selected from thegroup consisting of a water-soluble ethylenically unsaturated C₃-C₆carboxylic acid, an allylamine, an inorganic acid addition salt of anallylamine, a di-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylate, a methacrylate,an olefinically unsaturated nitrile, a diolefinically unsaturatedmonomer, N-vinyl pyrrolidone, N-vinyl formamide, an acrylamide, a loweralkyl-substituted acrylamide, a lower alkoxy-substituted acrylamide,N-vinylimidazole, N-vinylimidazoline, a styrene sulfonic acid and analkylene oxide.
 28. The water-dispersible nanoparticle of claim 27,wherein the hydrophilic regions each comprise at least one monomer unitselected from the group consisting of acrylic acid, methacrylic acid,styrene sulfonic acid, acrylamide and methacrylamide.
 29. Thewater-dispersible nanoparticle of claim 1, wherein the hydrophilicregions of the multiply amphipathic polymer each comprise a vinylmonomer substituted with at least one hydrophilic moiety selected fromthe group consisting of a carboxylate, a thiocarboxylate, an amide, animide, a hydrazine, a sulfonate, a sulfoxide, a sulfone, a sulfite, aphosphate, a phosphonate, a phosphonium, an alcohol, a thiol, a nitrate,an amine, an ammonium, and an alkyl ammonium group —[NHR¹R²]⁺, whereinR¹ and R² are alkyl substituents.
 30. The water-dispersible nanoparticleof claim 29, wherein the hydrophilic moiety is directly bound to acarbon atom in the polymer backbone of the multiply amphipathic polymer.31. The water-dispersible nanoparticle of claim 29, wherein thehydrophilic moiety is bound to a carbon atom in the backbone of themultiply amphipathic polymer through a linkage selected from the groupconsisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene,arylene, heteroarylene, alkarylene, and aralkylene.
 32. Thewater-dispersible nanoparticle of claim 1, wherein the multiplyamphipathic polymer is a copolymer of a hydrophilic monomer selectedfrom the group consisting of acrylic acid, methacrylic acid andcombinations thereof, with at least one hydrophobic alkyl (C₆-C₁₂)acrylamide monomer.
 33. The water-dispersible nanoparticle of claim 32,wherein the multiply amphipathic polymer is poly(acrylicacid-co-octylacrylamide).
 34. The water-dispersible nanoparticle ofclaim 1, wherein the multiply amphipathic polymer has a molecular weightin the range of approximately 500 to 50,000.
 35. The water-dispersiblenanoparticle of claim 34, wherein the multiply amphipathic polymer has amolecular weight in the range of approximately 1000 to 10,000.
 36. Thewater-dispersible nanoparticle of claim 35, wherein the multiplyamphipathic polymer has a molecular weight in the range of approximately1000 to
 5000. 37. The water-dispersible nanoparticle of claim 33,wherein the poly(acrylic acid-co-octylacrylamide) has a molecular weightin the range of approximately 1000 to
 5000. 38. The water-dispersiblenanoparticle of claim 1, wherein the hydrophobic regions represent inthe range of approximately 25 wt. % to 90 wt. % of the multiplyamphipathic polymer.
 39. The water-dispersible nanoparticle of claim 1,wherein the multiply amphipathic polymer is a polypeptide in which thehydrophobic regions are comprised of at least one hydrophobic amino acidand the hydrophilic regions are comprised of at least one hydrophilicamino acid.
 40. The water-dispersible nanoparticle of claim 1, whereinthe multiply amphipathic polymer is crosslinked.
 41. A compositioncomprising a plurality of water-dispersible nanoparticles eachcomprising: (a) an inner core comprised of a semiconductive or metallicmaterial; (b) a water-insoluble organic coating provided thereon; and(c) an outer layer of a multiply amphipathic polymer that comprises atleast two hydrophobic regions and at least two hydrophilic regions; and(d) a functional group covalently bound to the polymer through anoxyalkylene linking moiety.
 42. The composition of claim 41, wherein theinner cores of the nanoparticles are members of a monodisperse particlepopulation.
 43. The composition of claim 42, wherein the monodisperseparticle population is characterized in that when irradiated thepopulation emits light in a bandwidth in the range of approximately 20nm to 60 nm full width at half maximum (FWHM).
 44. The composition ofclaim 43, wherein the monodisperse particle population is characterizedin that when irradiated the population emits light in a bandwidth in therange of approximately 20 nm to 40 nm full width at half maximum (FWHM).45. The composition of claim 42, wherein the monodisperse particlepopulation is characterized in that it exhibits no more than about a 10%rms deviation in the diameter of the inner core.
 46. The composition ofclaim 45 wherein the monodisperse particle population is characterizedin that it exhibits no more than about a 5% rms deviation in thediameter of the inner core.
 47. The composition of claim 41, wherein theoxyalkylene linking moiety is composed of 2 to about 20 carbon atoms.48. The composition of claim 41, wherein the functional group isselected from amino, cyano, carboxyl, ester, and halocarbonyl groups.49. The composition of claim 48 wherein the functional group is an aminogroup.
 50. The composition of claim 41, wherein the inner core iscomprised of a semiconductive material.
 51. The composition of claim 50,wherein the semiconductive material is inorganic.
 52. The composition ofclaim 51, wherein the semiconductive material is crystalline.
 53. Thecomposition of claim 50, wherein the water-insoluble organic coating iscomprised of trioctylphosphine oxide, trioctylphosphine,tributylphosphine, or a mixture thereof.
 54. The composition of claim50, further including a shell layer between the core and thewater-insoluble organic coating.
 55. The composition of claim 54,wherein the shell layer is comprised of a semiconductive material havinga band gap energy greater than that of the inner core.
 56. Thecomposition of claim 41, wherein the inner core is comprised of ametallic material.
 57. The composition of claim 56, wherein thewater-insoluble organic coating has an affinity for the metallicmaterial.
 58. The composition of claim 57, wherein the water-insolubleorganic coating is comprised of a hydrophobic surfactant.
 59. Thecomposition of claim 58, wherein the hydrophobic surfactant is selectedfrom the group consisting of octanethiol, dodecanethiol, dodecylamine,tetraoctylammonium bromide, and mixtures thereof.
 60. The composition ofclaim 41, wherein the multiply amphipathic polymer is linear orbranched.
 61. The composition of claim 60, wherein the multiplyamphipathic polymer is branched.
 62. The composition of claim 61,wherein the multiply amphipathic polymer is hyperbranched or dendritic.63. The composition of claim 41, wherein the hydrophobic regions of themultiply amphipathic polymer are each comprised of at least onenon-ionizable, nonpolar monomer unit.
 64. The composition of claim 41,wherein each of the hydrophobic regions comprise at least one monomerunit selected from the group consisting of ethylene, propylene, alkyl(C₄-C₁₂)-substituted ethyleneimine, an alkyl acrylate, a methacrylate, aphenyl acrylate, an alkyl acrylamide, a styrene, a hydrophobicallyderivatized styrene, a vinyl ether, a vinyl ester, a vinyl halide, and acombination thereof.
 65. The composition of claim 58, wherein thehydrophobic regions comprise at least one monomer unit selected from analkyl acrylate, an alkyl methacrylate, an alkyl acrylamide, and amixture thereof.
 66. The composition of claim 41, wherein thehydrophilic regions of the multiply amphipathic polymer each comprise atleast one monomer unit containing an ionizable or polar moiety.
 67. Thecomposition of claim 66, wherein the hydrophilic regions each compriseat least one monomer unit containing an ionizable moiety.
 68. Thecomposition of claim 67, wherein the ionizable moiety is selected fromthe group consisting of carboxylic acid, sulfonic acid, phosphonic acid,and amine substituents.
 69. The composition of claim 63, wherein thehydrophilic regions each comprise at least one monomer unit containingan ionizable or polar moiety.
 70. The composition of claim 69, whereinthe hydrophilic regions each comprise at least one monomer unitcontaining an ionizable moiety.
 71. The composition of claim 70, whereinthe ionizable moiety is selected from the group consisting of carboxylicacid, sulfonic acid, phosphonic acid, and an amine substituent.
 72. Thecomposition of claim 41, wherein the hydrophilic regions of the multiplyamphipathic polymer each comprise at least one monomer unit selectedfrom the group consisting of a water-soluble ethylenically unsaturatedC₃-C₆ carboxylic acid, an allylamine, an inorganic acid addition salt ofan allylamine, a di-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylate, amethacrylate, an olefinically unsaturated nitrile, a diolefinicallyunsaturated monomer, N-vinyl pyrrolidone, N-vinyl formamide, anacrylamide, a lower alkyl-substituted acrylamide, a loweralkoxy-substituted acrylamide, N-vinylimidazole, N-vinylimidazoline, astyrene sulfonic acid and an alkylene oxide.
 73. The composition ofclaim 72, wherein the hydrophilic regions each comprise at least onemonomer unit selected from the group consisting of acrylic acid,methacrylic acid, styrene sulfonic acid, acrylamide and methacrylamide.74. The composition of claim 41, wherein the hydrophilic regions of themultiply amphipathic polymer each comprise a vinyl monomer substitutedwith at least one hydrophilic moiety selected from the group consistingof a carboxylate, a thiocarboxylate, an amide, an imide, a hydrazine, asulfonate, a sulfoxide, a sulfone, a sulfite, a phosphate, aphosphonate, a phosphonium, an alcohol, a thiol, a nitrate, an amine, ananunonium, and an alkyl ammonium group —[NHR¹R²]⁺, wherein R¹ and R² arealkyl substituents.
 75. The composition of claim 74, wherein thehydrophilic moiety is directly bound to a carbon atom in the backbone ofthe multiply amphipathic polymer.
 76. The composition of claim 74,wherein the hydrophilic moiety is bound to a carbon atom in the backboneof the multiply amphipathic polymer through a linkage selected from thegroup consisting of alkylene, alkenylene, heteroalkylene,heteroalkenylene, arylene, heteroarylene, alkarylene, and aralkylene.77. The composition of claim 41, wherein the multiply amphipathicpolymer is a copolymer of a hydrophilic monomer selected from the groupconsisting of acrylic acid, methacrylic acid and combinations thereof,with at least one hydrophobic alkyl (C₆-C₁₂) acrylamide monomer.
 78. Thecomposition of claim 71, wherein the multiply amphipathic polymer ispoly(acrylic acid-co-octylacrylamide).
 79. The composition of claim 41,wherein the multiply amphipathic polymer has a molecular weight in therange of approximately 500 to 50,000.
 80. The composition of claim 79,wherein the multiply amphipathic polymer has a molecular weight in therange of approximately 1000 to 10,000.
 81. The composition of claim 80,wherein the multiply amphipathic polymer has a molecular weight in therange of approximately 1000 to
 5000. 82. The composition of claim 78,wherein the poly(acrylic acid-co-octylacrylamide) has a molecular weightin the range of approximately 1000 to
 5000. 83. The composition of claim41, wherein the hydrophobic regions of the multiply amphipathic polymerrepresent in the range of approximately 25 wt. % to 90 wt. % of thepolymer.
 84. The composition of claim 41, wherein the multiplyamphipathic polymer is a polypeptide in which the hydrophobic regionsare comprised of at least one hydrophobic amino acid and the hydrophilicregions are comprised of at least one hydrophilic amino acid.
 85. Thecomposition of claim 41, wherein the multiply amphipathic polymer iscrosslinked.
 86. A conjugate of an affinity molecule and awater-dispersible nanoparticle, wherein the nanoparticle comprises (a)an inner core comprised of a semiconductive or metallic material, (b) awater-insoluble organic coating provided thereon, (c) an outer layer ofa multiply amphipathic polymer having at least two hydrophobic regionsand at least two hydrophilic regions, and (d) a functional groupcovalently linked to the polymer, and further wherein the affinitymolecule is bound to the functional group through an oxyalkylene linkingmoiety.
 87. The conjugate of claim 86, wherein the affinity molecule isselected so as to specifically bind to a biological target molecule. 88.The conjugate of claim 87, wherein the affinity molecule is selectedfrom the group consisting of an antigen, hapten, antibody, antibodyfragment, hormone, hormone binding protein, enzyme, enzyme inhibitor,nucleic acid, nucleotide, oligonucleotide, polynucleotide, receptoragonist, and receptor antagonist.
 89. The conjugate of claim 87, whereinthe affinity molecule specifically binds to the biological targetmolecule through noncovalent interaction.
 90. The conjugate of claim 89,wherein the affinity molecule specifically binds to the biologicaltarget molecule through noncovalent interaction.
 91. The conjugate ofclaim 86, wherein the oxyalkylene linking moiety is composed of 2 toabout 20 carbon atoms.
 92. The conjugate of claim 86, wherein thefunctional group is selected from amino, cyano, carboxyl, ester, andhalocarbonyl groups.
 93. The conjugate of claim 92, wherein thefunctional group is an amino group.
 94. The water-dispersiblenanoparticle of claim 86, wherein the inner core is comprised of asemiconductive material.
 95. The conjugate of claim 94, wherein thesemiconductive material is inorganic.
 96. The conjugate of claim 95,wherein the semiconductive material is crystalline.
 97. The conjugate ofclaim 94, wherein the water-insoluble organic coating is comprised oftrioctylphosphine oxide, trioctylphosphine, tributylphosphine, or amixture thereof.
 98. The conjugate of claim 94, further including ashell layer between the core and the water-insoluble organic coating.99. The conjugate of claim 98, wherein the shell layer is comprised of asemiconductive material having a band gap energy greater than that ofthe inner core.
 100. The conjugate of claim 86, wherein the inner coreis comprised of a metallic material.
 101. The conjugate of claim 100,wherein the water-insoluble organic coating has an affinity for themetallic material.
 102. The conjugate of claim 101, wherein thewater-insoluble organic coating is comprised of a hydrophobicsurfactant.
 103. The conjugate of claim 102, wherein the hydrophobicsurfactant is selected from the group consisting of octanethiol,dodecanethiol, dodecylamine, tetraoctylammonium bromide, and mixturesthereof.
 104. The conjugate of claim 86, wherein the multiplyamphipathic polymer is linear or branched.
 105. The conjugate of claim104, wherein the multiply amphipathic polymer is branched.
 106. Theconjugate of claim 105, wherein the multiply amphipathic polymer ishyperbranched or dendritic.
 107. The conjugate of claim 86, wherein thehydrophobic regions of the multiply amphipathic polymer are eachcomprised of at least one non-ionizable, nonpolar monomer unit.
 108. Theconjugate of claim 86, wherein each of the hydrophobic regions compriseat least one monomer unit selected from the group consisting ofethylene, propylene, alkyl (C₄-C₁₂)-substituted ethyleneimine, an alkylacrylate, a methacrylate, a phenyl acrylate, an alkyl acrylamide, astyrene, a hydrophobically derivatized styrene, a vinyl ether, a vinylester, a vinyl halide, and a combination thereof.
 109. The conjugate ofclaim 108, wherein the hydrophobic regions comprise at least one monomerunit selected from an alkyl acrylate, an alkyl methacrylate, an alkylacrylamide, and a mixture thereof.
 110. The conjugate of claim 86,wherein the hydrophilic regions of the multiply amphipathic polymer eachcomprise at least one monomer unit containing an ionizable or polarmoiety.
 111. The conjugate of claim 110, wherein the hydrophilic regionseach comprise at least one monomer unit containing an ionizable moiety.112. The conjugate of claim 111, wherein the ionizable moiety isselected from the group consisting of carboxylic acid, sulfonic acid,phosphonic acid, and amine substituents.
 113. The conjugate of claim107, wherein the hydrophilic regions each comprise at least one monomerunit containing an ionizable or polar moiety.
 114. The conjugate ofclaim 113, wherein the hydrophilic regions each comprise at least onemonomer unit containing an ionizable moiety.
 115. The conjugate of claim114, wherein the ionizable moiety is selected from the group consistingof carboxylic acid, sulfonic acid, phosphonic acid, and an aminesubstituent.
 116. The conjugate of claim 86, wherein the hydrophilicregions of the multiply amphipathic polymer each comprise at least onemonomer unit selected from the group consisting of a water-solubleethylenically unsaturated C₃-C₆ carboxylic acid, an allylamine, aninorganic acid addition salt of an allylamine, adi-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylate, a methacrylate, anolefinically unsaturated nitrile, a diolefinically unsaturated monomer,N-vinyl pyrrolidone, N-vinyl formamide, an acrylamide, a loweralkyl-substituted acrylamide, a lower alkoxy-substituted acrylamide,N-vinylimidazole, N-vinylimidazoline, a styrene sulfonic acid and analkylene oxide.
 117. The conjugate of claim 116, wherein the hydrophilicregions each comprise at least one monomer unit selected from the groupconsisting of acrylic acid, methacrylic acid, styrene sulfonic acid,acrylamide and methacrylamide.
 118. The conjugate of claim 86, whereinthe hydrophilic regions of the multiply amphipathic polymer eachcomprise a vinyl monomer substituted with at least one hydrophilicmoiety selected from the group consisting of a carboxylate, athiocarboxylate, an amide, an imide, a hydrazine, a sulfonate, asulfoxide, a sulfone, a sulfite, a phosphate, a phosphonate, aphosphonium, an alcohol, a thiol, a nitrate, an amine, an ammonium, andan alkyl ammonium group —[NHR¹R²]⁺, wherein R¹ and R² are alkylsubstituents.
 119. The conjugate of claim 118, wherein the hydrophilicmoiety is directly bound to a carbon atom in the backbone of themultiply amphipathic polymer.
 120. The conjugate of claim 118, whereinthe hydrophilic moiety is bound to a carbon atom in the backbone of themultiply amphipathic polymer through a linkage selected from the groupconsisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene,arylene, heteroarylene, alkarylene, and aralkylene.
 121. The conjugateof claim 86, wherein the multiply amphipathic polymer is a copolymer ofa hydrophilic monomer selected from the group consisting of acrylicacid, methacrylic acid and combinations thereof, with at least onehydrophobic alkyl (C₆-C₁₂) acrylamide monomer.
 122. The conjugate ofclaim 121, wherein the multiply amphipathic polymer is poly(acrylicacid-co-octylacrylamide).
 123. The conjugate of claim 86, wherein themultiply amphipathic polymer has a molecular weight in the range ofapproximately 500 to 50,000.
 124. The conjugate of claim 123, whereinthe multiply amphipathic polymer has a molecular weight in the range ofapproximately 1000 to 10,000.
 125. The conjugate of claim 124, whereinthe multiply amphipathic polymer has a molecular weight in the range ofapproximately 1000 to
 5000. 126. The conjugate of claim 122, wherein thepoly(acrylic acid-co-octylacrylamide) has a molecular weight in therange of approximately 1000 to
 5000. 127. The conjugate of claim 86,wherein the hydrophobic regions represent in the range of approximately25 wt. % to 90 wt. % of the multiply amphipathic polymer.
 128. Theconjugate of claim 86, wherein the multiply amphipathic polymer is apolypeptide in which the hydrophobic regions are comprised of at leastone hydrophobic amino acid and the hydrophilic regions are comprised ofat least one hydrophilic amino acid.
 129. The conjugate of claim 86,wherein the multiply amphipathic polymer is crosslinked.
 130. Acomposition comprising a plurality of nanoparticle conjugates eachcomposed of a water-dispersible nanoparticle and an affinity molecule,wherein each water-dispersible nanoparticle comprises (a) an inner corecomprised of a semiconductive or metallic material, (b) awater-insoluble organic coating provided thereon, (c) an outer layer ofa multiply amphipathic polymer having at least two hydrophobic regionsand at least two hydrophilic regions, and (d) a functional groupcovalently linked to the polymer, and further wherein the affinitymolecule is bound to the functional group through an oxyalkylene linkingmoiety.
 131. The composition of claim 130, wherein the inner cores ofthe nanoparticles are members of a monodisperse particle population.132. The composition of claim 131, wherein the monodisperse particlepopulation is characterized in that when irradiated the population emitslight in a bandwidth in the range of approximately 20 nm to 60 nm fullwidth at half maximum (FWHM).
 133. The composition of claim 132, whereinthe monodisperse particle population is characterized in that whenirradiated the population emits light in a bandwidth in the range ofapproximately 20 nm to 40 nm full width at half maximum (FWHM).
 134. Thecomposition of claim 131, wherein the monodisperse particle populationis characterized in that it exhibits no more than about a 10% rmsdeviation in the diameter of the inner core.
 135. The composition ofclaim 134, wherein the monodisperse particle population is characterizedin that it exhibits no more than about a 5% rms deviation in thediameter of the inner core.
 136. The composition of claim 130, whereinthe affinity molecule is selected so as to specifically bind to abiological target molecule.
 137. The composition of claim 136, whereinthe affinity molecule is selected from the group consisting of anantigen, hapten, antibody, antibody fragment, hormone, hormone bindingprotein, enzyme, enzyme inhibitor, nucleic acid, nucleotide,oligonucleotide, polynucleotide, receptor agonist, and receptorantagonist.
 138. The composition of claim 136, wherein the affinitymolecule specifically binds to the biological target molecule throughnoncovalent interaction.
 139. The composition of claim 138, wherein theaffinity molecule specifically binds to the biological target moleculethrough noncovalent interaction.
 140. The composition of claim 130,wherein the oxyalkylene linking moiety is composed of 2 to about 20carbon atoms.
 141. The composition of claim 130, wherein the functionalgroup is selected from amino, cyano, carboxyl, ester, and halocarbonylgroups.
 142. The composition of claim 143, wherein the functional groupis an amino group.
 143. The composition of claim 130, wherein the innercore is comprised of a semiconductive material.
 144. The composition ofclaim 143, wherein the semiconductive material is inorganic.
 145. Thecomposition of claim 144, wherein the semiconductive material iscrystalline.
 146. The composition of claim 143, wherein thewater-insoluble organic coating is comprised of trioctylphosphine oxide,trioctylphosphine, tributylphosphine, or a mixture thereof.
 147. Thecomposition of claim 143, further including a shell layer between thecore and the water-insoluble organic coating.
 148. The composition ofclaim 147, wherein the shell layer is comprised of a semiconductivematerial having a band gap energy greater than that of the inner core.149. The composition of claim 130, wherein the inner core is comprisedof a metallic material.
 150. The composition of claim 149, wherein thewater-insoluble organic coating has an affinity for the metallicmaterial.
 151. The composition of claim 150, wherein the water-insolubleorganic coating is comprised of a hydrophobic surfactant.
 152. Thecomposition of claim 151, wherein the hydrophobic surfactant is selectedfrom the group consisting of octanethiol, dodecanethiol, dodecylamine,tetraoctylammonium bromide, and mixtures thereof.
 153. The compositionof claim 130, wherein the multiply amphipathic polymer is linear orbranched.
 154. The composition of claim 143, wherein the polymer isbranched.
 155. The composition of claim 154, wherein the polymer ishyperbranched or dendritic.
 156. The composition of claim 130, whereinthe hydrophobic regions of the multiply amphipathic polymer are eachcomprised of at least one non-ionizable, nonpolar monomer unit.
 157. Thecomposition of claim 130, wherein each of the hydrophobic regionscomprise at least one monomer unit selected from the group consisting ofethylene, propylene, alkyl (C₄-C₁₂)-substituted ethyleneimine, an alkylacrylate, a methacrylate, a phenyl acrylate, an alkyl acrylamide, astyrene, a hydrophobically derivatized styrene, a vinyl ether, a vinylester, a vinyl halide, and a combination thereof.
 158. The compositionof claim 157, wherein the hydrophobic regions comprise at least onemonomer unit selected from an alkyl acrylate, an alkyl methacrylate, analkyl acrylamide, and a mixture thereof.
 159. The composition of claim130, wherein the hydrophilic regions of the multiply amphipathic polymereach comprise at least one monomer unit containing an ionizable or polarmoiety.
 160. The composition of claim 159, wherein the hydrophilicregions each comprise at least one monomer unit containing an ionizablemoiety.
 161. The composition of claim 160, wherein the ionizable moietyis selected from the group consisting of carboxylic acid, sulfonic acid,phosphonic acid, and amine substituents.
 162. The composition of claim156, wherein the hydrophilic regions each comprise at least one monomerunit containing an ionizable or polar moiety.
 163. The composition ofclaim 162, wherein the hydrophilic regions each comprise at least onemonomer unit containing an ionizable moiety.
 164. The composition ofclaim 163, wherein the ionizable moiety is selected from the groupconsisting of carboxylic acid, sulfonic acid, phosphonic acid, and anamine substituent.
 165. The composition of claim 130, wherein thehydrophilic regions of the multiply amphipathic polymer each comprise atleast one monomer unit selected from the group consisting of awater-soluble ethylenically unsaturated C₃-C₆ carboxylic acid, anallylamine, an inorganic acid addition salt of an allylamine, adi-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylate, a methacrylate, anolefinically unsaturated nitrile, a diolefinically unsaturated monomer,N-vinyl pyrrolidone, N-vinyl formamide, an acrylamide, a loweralkyl-substituted acrylamide, a lower alkoxy-substituted acrylamide,N-vinylimidazole, N-vinylimidazoline, a styrene sulfonic acid and analkylene oxide.
 166. The composition of claim 165, wherein thehydrophilic regions each comprise at least one monomer unit selectedfrom the group consisting of acrylic acid, methacrylic acid, styrenesulfonic acid, acrylamide and methacrylamide.
 167. The composition ofclaim 130, wherein the hydrophilic regions of the multiply amphipathicpolymer each comprise a vinyl monomer substituted with at least onehydrophilic moiety selected from the group consisting of a carboxylate,a thiocarboxylate, an amide, an imide, a hydrazine, a sulfonate, asulfoxide, a sulfone, a sulfite, a phosphate, a phosphonate, aphosphonium, an alcohol, a thiol, a nitrate, an amine, an ammonium, andan alkyl ammonium group —[NHR¹R²]⁺, wherein R¹ and R² are alkylsubstituents.
 168. The composition of claim 167, wherein the hydrophilicmoiety is directly bound to a carbon atom in the backbone of themultiply amphipathic polymer.
 169. The composition of claim 167, whereinthe hydrophilic moiety is bound to a carbon atom in the backbone of themultiply amphipathic polymer through a linkage selected from the groupconsisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene,arylene, heteroarylene, alkarylene, and aralkylene.
 170. The compositionof claim 130, wherein the multiply amphipathic polymer is a copolymer ofa hydrophilic monomer selected from the group consisting of acrylicacid, methacrylic acid and combinations thereof, with at least onehydrophobic alkyl (C₆-C₁₂) acrylamide monomer.
 171. The composition ofclaim 170, wherein the multiply amphipathic polymer is poly(acrylicacid-co-octylacrylamide).
 172. The composition of claim 130, wherein themultiply amphipathic polymer has a molecular weight in the range ofapproximately 500 to 50,000.
 173. The composition of claim 172, whereinthe multiply amphipathic polymer has a molecular weight in the range ofapproximately 1000 to 10,000.
 174. The composition of claim 173, whereinthe multiply amphipathic polymer has a molecular weight in the range ofapproximately 1000 to
 5000. 175. The composition of claim 171, whereinthe poly(acrylic acid-co-octylacrylamide) has a molecular weight in therange of approximately 1000 to
 5000. 176. The composition of claim 130,wherein the hydrophobic regions represent in the range of approximately25 wt. % to 90 wt. % of the multiply amphipathic polymer.
 177. Thecomposition of claim 130, wherein the multiply amphipathic polymer is apolypeptide in which the hydrophobic regions are comprised of at leastone hydrophobic amino acid and the hydrophilic regions are comprised ofat least one hydrophilic amino acid.
 178. The composition of claim 130,wherein the multiply amphipathic polymer is crosslinked.
 179. Ananoparticle conjugate comprising: (a) a water-dispersible nanoparticleand an affinity molecule that serves as a first member of a bindingpair, wherein the nanoparticle comprises (i) an inner core comprised ofa semiconductive or metallic material, (ii) a water-insoluble organiccoating provided thereon, (iii) an outer layer of a multiply amphipathicpolymer having at least two hydrophobic regions and at least twohydrophilic regions, and (iv) a functional group covalently linked tothe polymer, and further wherein the affinity molecule is bound to thefunctional group through an oxyalkylene linking moiety; and (b) a secondmember of the binding pair associated with the first member througheither covalent or noncovalent interaction.
 180. The conjugate of claim179, wherein the affinity molecule is selected from the group consistingof an antigen, hapten, antibody, antibody fragment, hormone, hormonebinding protein, enzyme, enzyme inhibitor, nucleic acid, nucleotide,oligonucleotide, polynucleotide, receptor agonist, and receptorantagonist.
 181. The conjugate of claim 179, wherein the first andsecond members of the binding pair are selected from the groupconsisting of: antigen and antibody; antigen and antibody fragment;hapten and antibody; hapten and antibody fragment; biotin and avidin;biotin and streptavidin; hormone and hormone binding protein; receptoragonist and receptor; receptor antagonist and receptor; IgG and proteinA; lectin and carbohydrate; enzyme and enzyme cofactor; enzyme andenzyme inhibitor; and complementary oligonucleotides.
 182. The conjugateof claim 179, wherein the second member of the binding pair isassociated with the first member of the binding pair through a covalentbond.
 183. The conjugate of claim 179, wherein the second member of thebinding pair is associated with the first member of the binding pairthrough a noncovalent bond.
 184. The conjugate of claim 179, wherein theoxyalkylene linking moiety is composed of 2 to about 20 carbon atoms.185. The conjugate of claim 179, wherein the functional group isselected from amino, cyano, carboxyl, ester, and halocarbonyl groups.186. The conjugate of claim 185, wherein the functional group is anamino group.
 187. The conjugate of claim 179, wherein the inner core iscomprised of a semiconductive material.
 188. The conjugate of claim 187,wherein the semiconductive material is inorganic.
 189. The conjugate ofclaim 188, wherein the semiconductive material is crystalline.
 190. Theconjugate of claim 187, wherein the water-insoluble organic coating iscomprised of trioctylphosphine oxide, trioctylphosphine,tributylphosphine, or a mixture thereof.
 191. The conjugate of claim187, further including a shell layer between the core and thewater-insoluble organic coating.
 192. The conjugate of claim 191,wherein the shell layer is comprised of a semiconductive material havinga band gap energy greater than that of the inner core.
 193. Theconjugate of claim 179, wherein the inner core is comprised of ametallic material.
 194. The conjugate of claim 193, wherein thewater-insoluble organic coating has an affinity for the metallicmaterial.
 195. The conjugate of claim 194, wherein the water-insolubleorganic coating is comprised of a hydrophobic surfactant.
 196. Theconjugate of claim 195, wherein the hydrophobic surfactant is selectedfrom the group consisting of octanethiol, dodecanethiol, dodecylamine,tetraoctylammonium bromide, and mixtures thereof.
 197. The conjugate ofclaim 179, wherein the multiply amphipathic polymer is linear orbranched.
 198. The conjugate of claim 197, wherein the multiplyamphipathic polymer is branched.
 199. The conjugate of claim 198,wherein the multiply amphipathic polymer is hyperbranched or dendritic.200. The conjugate of claim 179, wherein the hydrophobic regions of themultiply amphipathic polymer are each comprised of at least onenon-ionizable, nonpolar monomer unit.
 201. The conjugate of claim 179,wherein each of the hydrophobic regions comprise at least one monomerunit selected from the group consisting of ethylene, propylene, alkyl(C₄-C₁₂)-substituted ethyleneimine, an alkyl acrylate, a methacrylate, aphenyl acrylate, an alkyl acrylamide, a styrene, a hydrophobicallyderivatized styrene, a vinyl ether, a vinyl ester, a vinyl halide, and acombination thereof.
 202. The conjugate of claim 201, wherein thehydrophobic regions comprise at least one monomer unit selected from analkyl acrylate, an alkyl methacrylate, an alkyl acrylamide, and amixture thereof.
 203. The conjugate of claim 179, wherein thehydrophilic regions of the multiply amphipathic polymer each comprise atleast one monomer unit containing an ionizable or polar moiety.
 204. Theconjugate of claim 203, wherein the hydrophilic regions each comprise atleast one monomer unit containing an ionizable moiety.
 205. Theconjugate of claim 204, wherein the ionizable moiety is selected fromthe group consisting of carboxylic acid, sulfonic acid, phosphonic acid,and amine substituents.
 206. The conjugate of claim 200, wherein thehydrophilic regions each comprise at least one monomer unit containingan ionizable or polar moiety.
 207. The conjugate of claim 206, whereinthe hydrophilic regions each comprise at least one monomer unitcontaining an ionizable moiety.
 208. The conjugate of claim 207, whereinthe ionizable moiety is selected from the group consisting of carboxylicacid, sulfonic acid, phosphonic acid, and an amine substituent.
 209. Theconjugate of claim 179, wherein the hydrophilic regions of the multiplyamphipathic polymer each comprise at least one monomer unit selectedfrom the group consisting of a water-soluble ethylenically unsaturatedC₃-C₆ carboxylic acid, an allylamine, an inorganic acid addition salt ofan allylamine, a di-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylate, amethacrylate, an olefinically unsaturated nitrile, a diolefinicallyunsaturated monomer, N-vinyl pyrrolidone, N-vinyl formamide, anacrylamide, a lower alkyl-substituted acrylamide, a loweralkoxy-substituted acrylamide, N-vinylimidazole, N-vinylimidazoline, astyrene sulfonic acid and an alkylene oxide.
 210. The conjugate of claim209, wherein the hydrophilic regions each comprise at least one monomerunit selected from the group consisting of acrylic acid, methacrylicacid, styrene sulfonic acid, acrylamide and methacrylamide.
 211. Theconjugate of claim 179, wherein the hydrophilic regions of the multiplyamphipathic polymer each comprise a vinyl monomer substituted with atleast one hydrophilic moiety selected from the group consisting of acarboxylate, a thiocarboxylate, an amide, an imide, a hydrazine, asulfonate, a sulfoxide, a sulfone, a sulfite, a phosphate, aphosphonate, a phosphonium, an alcohol, a thiol, a nitrate, an amine, anammonium, and an alkyl ammonium group —[NHR¹R²]⁺, wherein R¹ and R² arealkyl substituents.
 212. The conjugate of claim 211, wherein thehydrophilic moiety is directly bound to a carbon atom in the backbone ofthe multiply amphipathic polymer.
 213. The conjugate of claim 211,wherein the hydrophilic moiety is bound to a carbon atom in the backboneof the multiply amphipathic polymer through a linkage selected from thegroup consisting of alkylene, alkenylene, heteroalkylene,heteroalkenylene, arylene, heteroarylene, alkarylene, and aralkylene.214. The conjugate of claim 179, wherein the multiply amphipathicpolymer is a copolymer of a hydrophilic monomer selected from the groupconsisting of acrylic acid, methacrylic acid and combinations thereof,with at least one hydrophobic alkyl (C₆-C₁₂) acrylamide monomer. 215.The conjugate of claim 214, wherein the multiply amphipathic polymer ispoly(acrylic acid-co-octylacrylamide).
 216. The conjugate of claim 179,wherein the multiply amphipathic polymer has a molecular weight in therange of approximately 500 to 50,000.
 217. The conjugate of claim 216,wherein the multiply amphipathic polymer has a molecular weight in therange of approximately 1000 to 10,000.
 218. The conjugate of claim 217,wherein the multiply amphipathic polymer has a molecular weight in therange of approximately 1000 to
 5000. 219. The conjugate of claim 215,wherein the poly(acrylic acid-co-octylacrylamide) has a molecular weightin the range of approximately 1000 to
 5000. 220. The conjugate of claim179, wherein the hydrophobic regions represent in the range ofapproximately 25 wt. % to 90 wt. % of the multiply amphipathic polymer.221. The conjugate of claim 179, wherein the multiply amphipathicpolymer is a polypeptide in which the hydrophobic regions are comprisedof at least one hydrophobic amino acid and the hydrophilic regions arecomprised of at least one hydrophilic amino acid.
 222. The conjugate ofclaim 179, wherein the multiply amphipathic polymer is crosslinked. 223.A composition comprising a plurality of nanoparticle conjugates eachcomprising: (a) a water-dispersible nanoparticle and an affinitymolecule that serves as a first member of a binding pair, wherein thenanoparticle comprises (i) an inner core comprised of a semiconductiveor metallic material, (ii) a water-insoluble organic coating providedthereon, (iii) an outer layer of a multiply amphipathic polymer havingat least two hydrophobic regions and at least two hydrophilic regions,and (iv) a functional group covalently linked to the polymer, andfurther wherein the affinity molecule is bound to the functional groupthrough an oxyalkylene linking moiety; and (b) a second member of thebinding pair associated with the first member through either covalent ornoncovalent interaction.
 224. The composition of claim 223, wherein theinner cores of the nanoparticles are members of a monodisperse particlepopulation.
 225. The composition of claim 224, wherein the monodisperseparticle population is characterized in that when irradiated thepopulation emits light in a bandwidth in the range of approximately 20nm to 60 nm full width at half maximum (FWHM).
 226. The composition ofclaim 225, wherein the monodisperse particle population is characterizedin that when irradiated the population emits light in a bandwidth in therange of approximately 20 nm to 40 nm full width at half maximum (FWHM).227. The composition of claim 224, wherein the monodisperse particlepopulation is characterized in that it exhibits no more than about a 10%rms deviation in the diameter of the inner core.
 228. The composition ofclaim 227, wherein the monodisperse particle population is characterizedin that it exhibits no more than about a 5% rms deviation in thediameter of the inner core.
 229. The composition of claim 223, whereinthe affinity molecule is selected from the group consisting of anantigen, hapten, antibody, antibody fragment, hormone, hormone bindingprotein, enzyme, enzyme inhibitor, nucleic acid, nucleotide,oligonucleotide, polynucleotide, receptor agonist, and receptorantagonist.
 230. The composition of claim 223, wherein the first andsecond members of the binding pair are selected from the groupconsisting of: antigen and antibody; antigen and antibody fragment;hapten and antibody; hapten and antibody fragment; biotin and avidin;biotin and streptavidin; hormone and hormone binding protein; receptoragonist and receptor; receptor antagonist and receptor; IgG and proteinA; lectin and carbohydrate; enzyme and enzyme cofactor; enzyme andenzyme inhibitor; and complementary oligonucleotides.
 231. Thecomposition of claim 223, wherein the second member of the binding pairis associated with the first member of the binding pair through acovalent bond.
 232. The composition of claim 223, wherein the secondmember of the binding pair is associated with the first member of thebinding pair through a noncovalent bond.
 233. The composition of claim223, wherein the oxyalkylene linking moiety is composed of 2 to about 20carbon atoms.
 234. The composition of claim 223, wherein the functionalgroup is selected from amino, cyano, carboxyl, ester, and halocarbonylgroups.
 235. The composition of claim 234, wherein the functional groupis an amino group.
 236. The composition of claim 223, wherein the innercore is comprised of a semiconductive material.
 237. The composition ofclaim 236, wherein the semiconductive material is inorganic.
 238. Thecomposition of claim 237, wherein the semiconductive material iscrystalline.
 239. The composition of claim 236, wherein thewater-insoluble organic coating is comprised of trioctylphosphine oxide,trioctylphosphine, tributylphosphine, or a mixture thereof.
 240. Thecomposition of claim 236, further including a shell layer between thecore and the water-insoluble organic coating.
 241. The composition ofclaim 240, wherein the shell layer is comprised of a semiconductivematerial having a band gap energy greater than that of the inner core.242. The composition of claim 223, wherein the inner core is comprisedof a metallic material.
 243. The composition of claim 242, wherein thewater-insoluble organic coating has an affinity for the metallicmaterial.
 244. The composition of claim 243, wherein the water-insolubleorganic coating is comprised of a hydrophobic surfactant.
 245. Thecomposition of claim 244, wherein the hydrophobic surfactant is selectedfrom the group consisting of octanethiol, dodecanethiol, dodecylamine,tetraoctylammonium bromide, and mixtures thereof.
 246. The compositionof claim 223, wherein the multiply amphipathic polymer is linear orbranched.
 247. The composition of claim 236, wherein the polymer isbranched.
 248. The composition of claim 247, wherein the polymer ishyperbranched or dendritic.
 249. The composition of claim 223, whereinthe hydrophobic regions of the multiply amphipathic polymer are eachcomprised of at least one non-ionizable, nonpolar monomer unit.
 250. Thecomposition of claim 223, wherein each of the hydrophobic regionscomprise at least one monomer unit selected from the group consisting ofethylene, propylene, alkyl (C₄-C₁₂)-substituted ethyleneimine, an alkylacrylate, a methacrylate, a phenyl acrylate, an alkyl acrylamide, astyrene, a hydrophobically derivatized styrene, a vinyl ether, a vinylester, a vinyl halide, and a combination thereof.
 251. The compositionof claim 250, wherein the hydrophobic regions comprise at least onemonomer unit selected from an alkyl acrylate, an alkyl methacrylate, analkyl acrylamide, and a mixture thereof.
 252. The composition of claim223, wherein the hydrophilic regions of the multiply amphipathic polymereach comprise at least one monomer unit containing an ionizable or polarmoiety.
 253. The composition of claim 252, wherein the hydrophilicregions each comprise at least one monomer unit containing an ionizablemoiety.
 254. The composition of claim 253, wherein the ionizable moietyis selected from the group consisting of carboxylic acid, sulfonic acid,phosphonic acid, and amine substituents.
 255. The composition of claim249, wherein the hydrophilic regions each comprise at least one monomerunit containing an ionizable or polar moiety.
 256. The composition ofclaim 255, wherein the hydrophilic regions each comprise at least onemonomer unit containing an ionizable moiety.
 257. The composition ofclaim 256, wherein the ionizable moiety is selected from the groupconsisting of carboxylic acid, sulfonic acid, phosphonic acid, and anamine substituent.
 258. The composition of claim 223, wherein thehydrophilic regions of the multiply amphipathic polymer each comprise atleast one monomer unit selected from the group consisting of awater-soluble ethylenically unsaturated C₃-C₆ carboxylic acid, anallylamine, an inorganic acid addition salt of an allylamine, adi-C₁-C₃-alkylamino-C₂-C₆-alkyl acrylate, a methacrylate, anolefinically unsaturated nitrile, a diolefinically unsaturated monomer,N-vinyl pyrrolidone, N-vinyl formamide, an acrylamide, a loweralkyl-substituted acrylamide, a lower alkoxy-substituted acrylamide,N-vinylimidazole, N-vinylimidazoline, a styrene sulfonic acid and analkylene oxide.
 259. The composition of claim 258, wherein thehydrophilic regions each comprise at least one monomer unit selectedfrom the group consisting of acrylic acid, methacrylic acid, styrenesulfonic acid, acrylamide and methacrylamide.
 260. The composition ofclaim 223, wherein the hydrophilic regions of the multiply amphipathicpolymer each comprise a vinyl monomer substituted with at least onehydrophilic moiety selected from the group consisting of a carboxylate,a thiocarboxylate, an amide, an imide, a hydrazine, a sulfonate, asulfoxide, a sulfone, a sulfite, a phosphate, a phosphonate, aphosphonium, an alcohol, a thiol, a nitrate, an amine, an ammonium, andan alkyl ammonium group —[NHR¹R²]⁺, wherein R¹ and R² are alkylsubstituents.
 261. The composition of claim 260, wherein the hydrophilicmoiety is directly bound to a carbon atom in the backbone of themultiply amphipathic polymer.
 262. The composition of claim 260, whereinthe hydrophilic moiety is bound to a carbon atom in the backbone of themultiply amphipathic polymer through a linkage selected from the groupconsisting of alkylene, alkenylene, heteroalkylene, heteroalkenylene,arylene, heteroarylene, alkarylene, and aralkylene.
 263. The compositionof claim 223, wherein the multiply amphipathic polymer is a copolymer ofa hydrophilic monomer selected from the group consisting of acrylicacid, methacrylic acid and combinations thereof, with at least onehydrophobic alkyl (C₆-C₁₂) acrylamide monomer.
 264. The composition ofclaim 263, wherein the multiply amphipathic polymer is poly(acrylicacid-co-octylacrylamide).
 265. The composition of claim 223, wherein themultiply amphipathic polymer has a molecular weight in the range ofapproximately 500 to 50,000.
 266. The composition of claim 265, whereinthe multiply amphipathic polymer has a molecular weight in the range ofapproximately 1000 to 10,000.
 267. The composition of claim 266, whereinthe multiply amphipathic polymer has a molecular weight in the range ofapproximately 1000 to
 5000. 268. The composition of claim 264, whereinthe poly(acrylic acid-co-octylacrylamide) has a molecular weight in therange of approximately 1000 to
 5000. 269. The composition of claim 223,wherein the hydrophobic regions represent in the range of approximately25 wt. % to 90 wt. % of the multiply amphipathic polymer.
 270. Thecomposition of claim 223, wherein the multiply amphipathic polymer is apolypeptide in which the hydrophobic regions are comprised of at leastone hydrophobic amino acid and the hydrophilic regions are comprised ofat least one hydrophilic amino acid.
 271. The composition of claim 223,wherein the multiply amphipathic polymer is crosslinked.